CN101490553A - Method of treating diseases with parp inhibitors - Google Patents

Abstract

The present invention relates to methods of identifying a disease treatable with PARP modulators by identifying a level of PARP in a sample of a subject, making a decision regarding identifying the disease treatable by the PARP modulators wherein the decision is made based on the level of PARP. The method further comprises of treating the disease in the subject with the PARP modulators. The methods relate to identifying up-regulated PARP in a disease and making a decision regarding the treatment of the disease with PARP inhibitors. The extent of PARP up-regulation in a disease can also help in determining the efficacy of the treatement with PARP inhibitors. The present invention discloses various diseases that have up-regulated or down-regulated PARP and can be treated with PARP inhibitors or PARP activators, respectively. The examples of the diseases include cancer, inflammation, metabolic disease, CVS disease, CNS disease, disorder of hematolymphoid system, disorder of endocrine and neuroendocrine, disorder of urinary tract, disorder of respiratory system, disorder of female reproductive system, and disorder of male reproductive system.

Description

Method with PARP inhibitor for treating disease

Related application

The U.S. Provisional Application 60/866602 that the U.S. Provisional Application 60/804563 that the application and on June 12nd, 2006 submit to and on November 20th, 2006 submit to is relevant, and the complete introducing of this paper is above-mentioned applies for reference.

Background of invention

PARP (poly-ADP ribose polymerase) participates in comprising the relevant function of multiple DNA of cell proliferation, differentiation, apoptosis, DNA reparation, also influence telomere length and chromosomal stability (d ' people such as Adda di Fagagna, 1999, Nature Gen., 23 (1): 76-80).The overactivity of the PARP that oxidative stress is induced consumes NAD+, and therefore consumes ATP, finally causes cell dysfunction or necrosis.The pathomechanism of various other forms of cardiovascular functional disorder, shock, traumatic central lesion, arthritis, colitis, allergic encephalitis and inflammation that this cell suicide mechanism is correlated with cancer, apoplexy, myocardial ischemia, diabetes, diabetes is relevant.Proved that also PARP is relevant with some transcription factors and regulated its function.The multi-functional of PARP makes it become the target of the multiple serious disease that comprises various types of cancers and neurodegenerative disease.

Breast cancer is the malignant tumour that is developed into by the cell in the breast.It is except cutaneum carcinoma in the women common cancer, and be the second largest reason of woman cancer associated death.The common overexpression HER2/neu of the breast cancer of tubercle positive oncogene, meaning at cell surface has than the more copy number of the normal quantity of HER2 protein.Breast cancer women diffusion with more HER2 gene copy numbers is the fastest, and has worse prognosis.This breast cancer subgroup is general with the Her-2 Antybody therapy that is called Herceptin (Trastuzumab).

The probability that develops into breast cancer when carrying the women to 70 year old of non-functional BRCA1 and BRCA2 gene and molecular pathways thereof can reach 85%.Conclusion according to breast cancer associating group (Breast CancerLinkage Consortium) (1997), the histology of easily suffering from the women of breast cancer owing to carry BRCA1 and BRCA2 (600185) sudden change is different from Sporadic cases, and between the breast cancer of BRCA1 and BRCA2 carriers of mutation difference is arranged also.

The PARP inhibitor can effectively kill and wound the tumour cell in the defective individuality in BRCA1 and BRCA2 (Byrant waits the people, 2005, Nature, 434 (7035): 913-7 and Farmer, wait the people, 2005, Nature, 434 (7035): 917-21).The PARP inhibitor has help has the particular patient subgroup of sudden change in these genes potentiality.These sudden changes make the patient tend to take place in early days cancer, and find in breast cancer, oophoroma, prostate cancer and cancer of pancreas.Existing early detection strategy means that the medical professional will just find them in the very early stage stage of cancer, and this moment, they were treatments easily.For example, the simple screening technique that is called as colonoscopy can be found polyp before polyp has the possibility that canceration takes place.But more effective and strong early diagnosis of cancer strategy is extremely useful for prevention with more effectively treating cancer.

Summary of the invention

On the one hand, the invention provides by measuring PARP level among the experimenter and determine that experimenter's disease can use the method for PARP inhibitor for treating, if and the PARP among the experimenter then further provides the treatment of using PARP inhibitor itself or uniting with other medicines or treatment to the experimenter to adjusted.

An aspect of of the present present invention relates to a kind of definite disease or disease stage can be with the method for PARP modulators for treatment, comprise the PARP level in the sample of determining the experimenter, make the decision that to use the PARP modulators for treatment about definite this disease, wherein, this decision is to make according to the expression of PARP.Some preferred embodiment in, the level of PARP is to adjusted.An aspect of of the present present invention relates to a kind of definite disease or disease stage can be united the method that other medicines are treated with the PARP correctives, comprise the PARP level in the sample of determining the experimenter, make decision about determining that this disease can use PARP correctives associating other medicines to treat, wherein, this decision is to make according to the expression of PARP.Some preferred embodiment in, the level of PARP is to adjusted.

Another aspect of the present invention relates to a kind of method of the disease by PARP modulators for treatment experimenter, comprise the PARP level in the sample of determining the experimenter, make about determining that this disease can be with the decision of PARP modulators for treatment according to the level of PARP, and with PARP modulators for treatment experimenter's disease.Some preferred embodiment in, the level of PARP is to adjusted.

In some embodiments, described disease is selected from cancer, inflammation, metabolic disease, CVS disease, CNS disease, blood-lymphatic system obstacle, endocrine and neuroendocrine obstacle, urethra obstacle, respiratory system obstacle, female reproductive system obstacle and male reproductive system obstacle.Some preferred embodiment in, described cancer is selected from adenocarcinoma of colon, adenocarcinoma of esophagus, hepatocellular carcinoma, squamous cell carcinoma, pancreas adenocarcinoma, islet-cell tumour, rectal adenocarcinoma, GISTs, sdenocarcinoma of stomach, adrenocortical carcinoma, follicular carcinoma, papillary carcinoma, breast cancer, duct carcinoma, lobular carcinoma, comedocarcinoma, mucous carcinoma, phyllodes tumor, adenocarcinoma ovaries, adenocarcinoma of endometrium, GCT, MCAC, adenocarcinoma of cervix, the vulva squamous cell carcinoma, basal-cell carcinoma, adenocarcinoma of the prostate, giant cell tumor of bone, the bone osteosarcoma, laryngocarcinoma, adenocarcinoma of lung, kidney, carcinoma of urinary bladder, wilms' tumor and lymthoma.

Some preferred embodiment in, described inflammation is selected from Wegner's granulomatosis, Hashimoto thyroiditis, hepatocellular carcinoma, chronic pancreatitis, rheumatoid arthritis, reactive lymphoid hyperplasia, osteoarthritis, ulcerative colitis and papillary carcinoma.Some preferred embodiment in, described metabolic disease is diabetes or obesity.Some preferred embodiment in, described CVS disease is selected from atherosclerotic, coronary artery disease, granulomatous myocarditis, chronic myocarditis, miocardial infarction and primary hypertrophic cardiomyopathy.Some preferred embodiment in, described CNS disease is selected from Alzheimer disease, cocaine abuse, schizophrenia and Parkinson's.Some preferred embodiment in, described blood-lymphatic system obstacle is selected from non_hodgkin lymphoma, chronic lymphocytic leukemia and reactive lymphoid hyperplasia.

Some preferred embodiment in, described endocrine and neuroendocrine obstacle are selected from nodular hyperplasia, Hashimoto thyroiditis, islet-cell tumour and papillary carcinoma.Some preferred embodiment in, described urethra obstacle is selected from clear-cell carcinoma, transitional cell carcinoma and wilms' tumor.Some preferred embodiment in, described respiratory system obstacle is selected from gland cancer, gland carcinoma squamosum, squamous cell carcinoma and large cell carcinoma.Some preferred embodiment in, described female reproductive system obstacle is selected from gland cancer, liomyoma, MCAC and serous cystadenocarcinoma.Some preferred embodiment in, described male reproductive system obstacle is selected from prostate cancer, benign protuberance hyperplasia and seminoma.

In some embodiments, the PARP level determines to comprise analytical technology.Some preferred embodiment in, this analytical technology is measured the PARP expression of gene.In some embodiments, sample is selected from people's normal specimens, tumor sample, hair, blood, cell, tissue, organ, brain tissue, blood, serum, sputum, saliva, blood plasma, nipple aspirated liquid (nipple aspirant), synovia, celiolymph, sweat, urine, fecal matter, pancreatic juice, girder liquid (trabecular fluid), celiolymph, tear, bronchial perfusate, swabbing (swabbings), bronchus aspirated liquid (bronchial aspirant), seminal fluid, prostatic fluid, level in gingival sulcus fluid (precervicular fluid), liquid (pre-ejaculate) before vaginal fluids and the ejaculation.Some preferred embodiment in, the level of PARP is to adjusted.In some embodiments, the level of PARP is to regulate downwards.In some embodiments, the PARP correctives is PARP inhibitor or antagonist.In some embodiments, PARP inhibitor or antagonist are selected from benzamide, quinolone, isoquinolone, benzopyrone, 3,5-two iodo-4-(4 '-the methoxyl phenoxy group) methyl benzoate and 3,5-two iodo-4-(4 '-methoxyl-3 ', 5 '-two iodo-phenoxy groups) and methyl benzoate, cyclohexyl benzamide, benzimidazole and indoles.

In some embodiments, this method further comprises to patient, health care supplier or health care administration person provides conclusion about disease, and this conclusion is based on above-mentioned decision.In some embodiments, treatment be selected from oral administration, mucosal, oral administration, intranasal administration, suction, parenteral (parental administration), intravenous, subcutaneous, the muscle, hypogloeeis, percutaneous dosing and rectally.

Another aspect of the present invention relates to the computer-readable medium that is suitable for transmitting the sample analysis result, wherein this medium comprises the information by the medicable disease of PARP correctives about the experimenter, described information obtains by following steps: determine the PARP level in experimenter's the sample, and make about the decision with PARP modulators for treatment disease according to the PARP level.In some embodiments, at least one step in this method uses a computer and finishes.

Another aspect of the present invention is related to the patient that the PARP inhibitor for treating is selected triple feminine genders (acceptor that lacks estrogen (ER-feminine gender) and progestational hormone (PR-feminine gender) and HER2 protein).In one embodiment, the cancer types with the PARP inhibitor for treating lacks estrogenic acceptor (ER-feminine gender).In another embodiment, the acceptor (PR-feminine gender) that lacks progestational hormone with the cancer types of PARP inhibitor for treating.In another kind of embodiment again, lack HER2 protein with the cancer types of PARP inhibitor for treating.

Another aspect of the present invention relates to the patient who selects the BRCA dependent pathway to have defective and organizes and with the PARP inhibitor it is treated.

Of the present invention relate to a kind of definite breast cancer more on the other hand can be with the method for PARP inhibitor or PARP antagonist for treating, comprise the PARP level in the sample of determining the experimenter, and make the decision that to use PARP inhibitor or PARP antagonist for treating about definite this breast cancer according to the PARP level.The method that relates to a kind of usefulness PARP inhibitor or PARP antagonist for treating experimenter's breast cancer more on the other hand of the present invention, comprise the PARP level in the sample of determining the experimenter, make about determining that this breast cancer can be with the decision of PARP modulators for treatment according to the PARP level, and with PARP inhibitor or PARP antagonist for treating breast cancer.In some embodiments, the level of PARP is to adjusted.In some embodiments, there is defective in experimenter's BRCA gene.In some embodiments, the experimenter has the BRCA gene of downward adjusting.In some method, the raising of PARP level is the indication of BRCA1 and/or BRACA2 defective.

On the one hand for diagnosing and/or treat the method for breast cancer.A kind of embodiment is to determine that breast cancer can comprise the PARP level in the sample of determining the experimenter with the method for PARP inhibitor for treating, and makes about the whether decision of available described PARP inhibitor for treating of described breast cancer according to described PARP level.Another kind of embodiment is the method with PARP inhibitor for treating experimenter's breast cancer, comprise the PARP level in the sample of determining described experimenter, make about the whether decision of available described PARP inhibitor for treating of described breast cancer according to described PARP level, and with the described breast cancer of described PARP inhibitor for treating.Another embodiment is the method to experimenter's breast cancer classification again, comprise the PARP level in the tumor sample of determining described experimenter, and make about the decision with the described tumour of PARP modulators for treatment, wherein, described decision is made according to described PARP level.Another kind of embodiment is the method for treatment experimenter's breast cancer, comprise the PARP level in the sample of determining described experimenter, make about decision according to described PARP level with PARP modulators for treatment described tumour, and with the described experimenter's of described PARP modulators for treatment described tumour.Breast cancer is preferably infitrating ductal carcinoma.In some embodiments, cancer is ER, Her2-neu and/or PR feminine gender.Another kind of embodiment is treatment experimenter's a method for cancer, comprise in the cancer sample of determining described experimenter and whether have ER, Her2-neu and PR, and with the described cancer of PARP inhibitor for treating, wherein, if described cancer sample is ER, Her2-neu and/or PR feminine gender, then carry out described treatment.

On the other hand, the method for diagnosis and/or treatment breast cancer comprises the experimenter's who relatively needs to diagnose or treat PARP level and the PARP level that presets.A kind of embodiment is the method that definite breast cancer can be used the PARP inhibitor for treating, comprise the PARP level in the sample of determining the experimenter, and determine that described PARP level whether on the level that presets, determines that thus described breast cancer is medicable with the PARP correctives.Another kind of embodiment is the method with PARP inhibitor for treating patient's breast cancer, comprise the PARP level in the sample of determining described patient, determine that described PARP level is whether on the level that presets, determine that thus described breast cancer is medicable with the PARP inhibitor, and by use the described breast cancer of described PARP inhibitor for treating to described patient.Typically, the experimenter also is BRCA1 or BRCA2 defective.Some experimenter has the BRCA expression of gene level of reduction.Another kind of embodiment is the method to patient's breast cancer classification, comprise the PARP level in the tumor sample of determining described patient, and determine described PARP level whether on the level that presets, thus with described breast cancer classification for medicable with the PARP correctives.A kind of method is the method for treatment experimenter's breast cancer, comprise the PARP level in the sample of determining described experimenter, determine that described PARP level is whether on the level that presets, determine that thus described breast cancer is medicable with the PARP correctives, and with the described patient's of described PARP modulators for treatment described tumour.Another method is the method that definite breast cancer can be used the PARP inhibitor for treating again, comprise the PARP level in the sample of determining the patient, determine that described PARP level whether on the level that presets, determines that thus described breast cancer is medicable with the PARP inhibitor.Another kind method is the method with PARP inhibitor for treating patient's breast cancer, comprise the PARP level in the sample of determining described patient, determine that described PARP level is whether on the level that presets, determine that thus described breast cancer is medicable with the PARP inhibitor, and by use the described breast cancer of described PARP inhibitor for treating to described patient.Typically, breast cancer is infitrating ductal carcinoma.Some infitrating ductal carcinoma is ER, Her2-neu and/or PR feminine gender.A kind of preferable methods is treatment patient's a method for cancer, comprise in the cancer sample of determining described patient and whether have ER, Her2-neu and/or PR, and when not having ER, Her2-neu and/or PR in described patient's the described sample, with the described cancer of PARP inhibitor for treating.

A kind of embodiment is the method that a certain stage of the disease of definite PARP mediation or the disease that PARP mediates can use the PARP modulators for treatment, comprise the PARP level in the sample of determining the experimenter, and determine that described PARP level whether on the level that presets, determines that thus the disease of described PARP mediation can be used the PARP modulators for treatment.Another kind of embodiment is by use the method for PARP modulators for treatment disease to the patient, comprise the PARP level in the sample of determining described patient, determine that described PARP level is whether on the level that presets, the disease of definite thus described PARP mediation can be used the PARP modulators for treatment, and by use the described patient's of described PARP modulators for treatment described disease to described patient.

An aspect of of the present present invention is the computer-readable medium that is suitable for transmitting the sample analysis result, wherein this medium comprises the information by the medicable disease of PARP correctives about the experimenter, described information obtains by following steps: determine the PARP level in described experimenter's the described sample, and determine that described PARP level whether on the level that presets, determines that thus the disease of described PARP mediation can be used the PARP modulators for treatment.

Other again one side of the present invention is to patient's group classification, and assessment is for the response of PARP treatment.A kind of embodiment is a method of selecting the experimenter for the PARP inhibitor for treating, be included in and use before the PARP inhibitor, the PARP level of mensuration from the biological sample that the experimenter collects determined that the PARP level in the sample is higher than preset value, and selected this experimenter to carry out the PARP inhibitor for treating.Another embodiment is the method with PARP inhibitor for treating experimenter again, be included in and use before the PARP inhibitor, the PARP level of mensuration from the biological sample that the experimenter collects determined that the PARP level in the sample is higher than preset value, and used the PARP inhibitor to this experimenter.Another kind of embodiment is that the method for the experimenter of PARP inhibitor for treating for the response of treatment accepted in evaluation, this method comprises: at least in first time point and the second time point determining experimenter's PARP level, to produce a PARP level and the 2nd PARP level at least, wherein, compare the positive response of the reduction indication of the 2nd PARP level with a PARP level for treatment.Typically, very first time point is before beginning with the PARP inhibitor for treating, and second time point is after beginning with the PARP inhibitor for treating.In some embodiments, very first time point is after beginning with the PARP inhibitor for treating, and the more late time point of second time point after very first time point, after a few days, a few weeks or months.Another kind of embodiment is the method that its situation of treatment causes the patient of PARP level rising, and wherein, the PARP level in patient's sample is higher than default PARP level, and this method comprises the PARP inhibitor of administering therapeutic effective dose.

The introducing of list of references

This paper is introduced in all publications of mentioning in the instructions and patented claim as a reference, as each independent publication or patented claim especially, be incorporated herein by reference individually.

The accompanying drawing summary

In appending claims, specifically illustrated new feature of the present invention.By with reference to following description the detailed Description Of The Invention of illustrative embodiments and accompanying drawing can obtain better understanding for feature of the present invention and advantage, in these embodiments, utilized principle of the present invention, accompanying drawing comprises:

Fig. 1 is the process flow diagram of the step of demonstration method disclosed herein.

Fig. 2 shows the computing machine of implementing the selected operation relevant with method disclosed herein.

Fig. 3 has shown the low correlativity of expressing of high expressed and BRCA1 and the BRCA2 of PARP1 in primary carcinoma of ovary.

Figure 4 and 5 have shown the expression of PARP in the infitrating ductal carcinoma hypotype.

Fig. 6 has shown the expression of PARP in pernicious ovary tissue and normal ovarian tissue.

Fig. 7 has shown the expression of PARP in pernicious endometrial tissue and normal endometrial tissue.

Fig. 8 has shown the expression of PARP in malign lung tissue and normal lung tissue.

Fig. 9 has shown the expression of PARP in malignant prostate tissue and normal prostate tissue.

Detailed Description Of The Invention

The grammer distortion of term " inhibition " or its same meaning as " inhibition ", does not require that the PARP activity reduces fully.Under the situation that lacks depression effect, for example, under the situation that lacks inhibitor such as PARP inhibitor disclosed by the invention, this reduction has preferably reduced about at least 50%, about at least 75%, about at least 90% and more preferably about at least 95% of molecular activity.Most preferably, this term refers to active observing or measurable reduction.Under the situation of treatment, suppress preferably enough to produce treatment of conditions and/or prevention benefit to being treated.

The grammer distortion of term used herein " sample ", " biological sample " or its same meaning refers to material known or that suspection is expressed the PARP level.Specimen can directly be used after the acquisition from the source or carry out using after the characteristic of pre-service with the change sample.Sample can as tissue or extract, comprise cell from any biogenetic derivation, and physiological fluid, for example, and whole blood, blood plasma, serum, saliva, eyepiece liquid, celiolymph, sweat, urine, milk, ascites, synovia, peritoneal fluid etc.Sample can obtain from the animal or human, preferably obtains from the people.Sample can be handled before use, for example prepares blood plasma, dilution viscous liquid etc. from blood.The method of handling sample comprises filtrations, distillation, extraction, concentrated, deactivation interference composition, adding reagent etc.

The grammer deformed finger of term used herein " experimenter " or its same meaning is health or that suffer from disease or suspect the warm-blooded animal that suffers from disease, as mammal.Preferably, " experimenter " refers to the people.

The grammer distortion of term used herein " treatment " or its same meaning means to obtain therapeutic benefit and/or preventative benefit.The therapeutic benefit is meant the elimination or the improvement of the illness of being treated.In addition, one or more with the relevant physiological signs of ill disease elimination or take a turn for the better and also can obtain the therapeutic benefit so that in the patient, observe improvement, although the patient may still suffer from described illness.For preventative benefit, can use composition to the patient in being in the danger that specified disease takes place or to the patient that report has one or more physiological signs of disease, even although may still not diagnose to this disease.

The stage of determining disease or disease can be with the method for PARP modulators for treatment

An aspect of of the present present invention, this method comprises determines that a kind of disease can comprise the PARP level in the sample of determining the experimenter by the PARP modulators for treatment, makes about determining that this disease can be by the decision of PARP modulators for treatment, wherein, this decision is to make according to the level of PARP.Another aspect of the present invention, this method comprises the disease with PARP modulators for treatment experimenter, comprise the PARP level in the sample of determining the experimenter, make about determining that this disease can be by the decision of PARP modulators for treatment according to the level of PARP, and with PARP modulators for treatment experimenter's disease.Another aspect of the present invention, this method further comprise to patient, health care supplier or health care administration person provides conclusion about disease, and wherein, this conclusion is based on above-mentioned decision.In some preferred implementation, disease is a breast cancer.In some preferred implementation, the level of PARP is to adjusted.In some preferred implementation, by measuring the level that the PARP expression of gene detects PARP.

The present invention relates to determine to suffer from the PARP level in a kind of experimenter's the sample of disease, wherein, when the level of PARP is during to adjusted, with PARP inhibitor or this experimenter of PARP antagonist for treating.The present invention has determined following disease, as cancer, inflammation, metabolic disease, CVS disease, CNS disease, blood-lymphatic system obstacle, endocrine and neuroendocrine obstacle, urethra obstacle, respiratory system obstacle, female reproductive system obstacle and male reproductive system obstacle, the level of PARP is to adjusted in these diseases.Therefore, the present invention determines that these diseases are available PARP inhibitor for treating.A kind of preferred embodiment in, the PARP inhibitor that is used for method of the present invention is the PARP-1 inhibitor.Being used for PARP inhibitor of the present invention can be by directly or indirectly interacting and plays a role with PARP, preferred PARP-1.PARP inhibitor used herein can be regulated one or more entities in PARP or the PARP approach.In some embodiments, the PARP inhibitor can suppress the PARP activity.

This method is being particularly useful aspect the treatment female reproductive system cancer.Have in heredity that breast cancer takes place among the women of BRCA1 or BRCA2 gene defect is because tumour cell has been lost the specific mechanism of repairing the DNA of damage.BRCA1 and BRCA2 are important for the dna double bound rupture reparation of being undertaken by homologous recombination, and the sudden change in these genes makes easily suffer from breast cancer and other cancer.PARP participates in the base excision and repairs, and this is the approach that the dna single bound rupture is repaired.The dysfunction of BRCA1 or BRCA2 makes cell for the inhibition sensitivity of PARP enzymatic activity, causes chromosome instability, cell cycle arrest and apoptosis subsequently.

The PARP inhibitor kills and wounds the cell of the DNA reparation that lacks this form, so the PARP inhibitor is being effective aspect the tumour cell that kills and wounds the BRCA defective and other the similar tumour cell.Normal cell can be by this drug influence, because they still have this DNA repair mechanism.This treatment is applicable to that also its behavior shows the breast cancer of other form of the tumour that is similar to the BRCA defective.Usually, use the medicine of not only killing tumor cells but also injuring normal cell to treat the patient with breast cancer.This has damage for normal cell, thereby causes distressful spinoff, as feeling sick and alopecia.In some embodiments, be that with an advantage of PARP inhibitor for treating it is a target: killing tumor cell, and as if normal cell unaffected.This is because the PARP inhibitor has used the special genes of some tumour cell to form.

The experimenter who the invention discloses the BRCA gene defect has the level to the PARP of adjusted.Fig. 3 has shown the low correlativity of expressing of high expressed and BRCA1 and the BRCA2 of PARP-1 in primary carcinoma of ovary.PARP's can be the indication that other DNA repairs the BRCA sample gene defect that pathway deficiency Buddhist monk do not recognize to adjusted.The evaluation of the gene expression of PARP-1 is the indication of tumour for the susceptibility of PARP inhibitor.Therefore, the invention provides by measuring the PARP level and determine the method for cancer early onset thereof among the patient of BRCA defective.If PARP is to adjusted, the patient that can determine the BRCA defective is medicable with the PARP inhibitor.Further, the patient of such BRCA defective can use the PARP inhibitor for treating.

The step of some method for optimizing of the present invention as shown in Figure 1.Scope of the present invention without limits, these steps can be to carry out independently of one another or one after the other.Can skip one or more steps in the method for the invention.In step 101, collect sample from the patient who suffers from disease.A kind of preferred embodiment in, sample is people's normal specimens and a tumor sample, hair, blood and other biofluid.In step 102, analyze the PARP level by the technology of knowing in this area, and, in step 103,, for example,, determine that this disease is medicable by the PARP inhibitor when PARP is during to adjusted according to the PARP level.Step 104 comprises the patient who suffers from disease with the PARP inhibitor for treating.Be appreciated that other method that this paper does not clearly propose that the present invention includes.Scope of the present invention without limits, the analysis of PARP and be known in the art in sample collection, the sample with other technology of PARP inhibitor for treating disease, and within the scope of the present invention.

In one embodiment of the invention, determine the tumour of homologous recombination defective by the expression of estimating PARP.If observe PARP to adjusted, then can use the such tumour of PARP inhibitor for treating.Another kind of embodiment is the method for cancer of treatment homologous recombination defective, comprises the expression of estimating PARP, and if observe overexpression, then use PARP inhibitor for treating cancer.

The collection of sample, preparation and separate

Biological sample of the present invention can obtain from the individuality of various states with different phenotype states such as cancer or other disease.The example of phenotype state also comprises normal subjects's phenotype, and it can be used for and ill experimenter's comparison.In some embodiments, suffering from the experimenter of disease is complementary with the control sample that the experimenter who never shows disease obtains.

Can collect sample from various sources, preferred human from mammal, comprise humoral sample or tissue sample.The sample of collecting can be people's normal specimens and tumor sample, hair, blood, other biofluid, cell, tissue, organ or body fluid, such as but not limited to, brain tissue, blood, serum, the sputum that comprises saliva, blood plasma, nipple aspirated liquid, synovia, celiolymph, sweat, urine, fecal matter, pancreatic juice, girder liquid, celiolymph, tear, bronchial perfusate, swabbing, bronchus aspirated liquid, seminal fluid, prostatic fluid, level in gingival sulcus fluid, vaginal fluids, the preceding liquid of ejaculation etc.Suitable tissue sample comprises various types of tumours or cancer tissue or organ-tissue, as those tissues that take out in biopsy.

Can be in one vertical period (for example, approximately once a day, weekly, every month once, every half a year once or once a year) repeatedly collect sample from individuality.Obtain from individuality in one period that many samples can be used for verifying the early detection result and/or the variation of the biological pattern for example determining to cause owing to progression of disease, drug therapy etc.

The preparation of sample can comprise any step with separating, and this depends on the analysis of the sample type and/or the PARP of collection.Only be as example, that such step comprises is concentrated, dilute, adjust pH, remove high-load polypeptide (for example, albumin, gamma Globulin and transferrins etc.), add the extraction and the purifying of the concentrating of antiseptic and caliberator, adding protease inhibitors, the desalination that adds denaturant, sample, sample protein matter, lipid.

The preparation of sample also can be separated in the molecule that is combined in the non-covalent bond compound on other protein (for example, carrier protein).This method can be separated those, and (the more common method of for example, albumin) molecule, or use as by for example using the molecule of sour protein denaturation from all carrier protein release combinations, is removed carrier protein then in conjunction with specific support protein.

Can use high-affinity reagent, high molecular filtrator, supercentrifugation and/or electrodialysis realize from sample, removing undesired protein (for example, high-load, information is not provided or detect less than protein).High-affinity reagent comprises antibody or optionally in conjunction with other reagent (for example, fit (aptamers)) of the protein of high-load.The preparation of sample also can comprise ion-exchange chromatography, metallic ion affinity chromatography, gel filtration, hydrophobic chromatography, chromatofocusing method, adsorption charomatography, isoelectric focusing and correlation technique.The molecular weight filtration device comprises the film based on size and molecular weight isolated molecule.Inverse osmosis, nanofiltration, ultrafiltration and micro-filtration can be further adopted in such filtration.

Supercentrifugation is a kind of method of removing undesired polypeptide from sample.Ultracentrifugation be with about 15000-60000 rev/min centrifugal to sample, use the precipitation (or its shortage) of optics system monitoring particulate simultaneously.Electrodialysis is a method of using electrolemma or semi-permeable diaphragm during the course, and wherein, ion is transferred to another kind of solution by semi-permeable diaphragm from a kind of solution under the influence of electric potential gradient.Because the film that uses in electrodialysis has following ability: selective transport has the ion of positive charge or negative charge, repel the ion that has opposite charges, or allow ionic species to move by semi-permeable diaphragm according to size and electric charge, so to concentrate, remove or separate be useful for electrolytical in electrodialysis.

Separation of the present invention and purifying can comprise any method known in the art, as Capillary Electrophoresis (for example, in kapillary or on chip) or chromatography (for example, in kapillary, in the post or on chip).Electrophoresis is a kind of method that is used in isolating ions molecule under the electric field effects.Electrophoresis can be in gel, in the kapillary or carry out in the microchannel on chip.The example that is used for the gel of electrophoresis comprises starch, acrylamide, polyoxyethylene, agarose or its combination.Gel can immobilization and adding pH gradient crosslinked by it, that add detergent or denaturant, enzyme or antibody (affinity electrophoresis) or substrate (zymography) be modified.The example capillaceous that is used for electrophoresis comprises the kapillary that is connected with the electrojet device.

Capillary Electrophoresis (CE) is preferred for separating the solute of composite hydrophilic molecule and altitudinal belt electric charge.The CE technology also can realize on micro-fluid chip.The type that depends on used kapillary and damping fluid, CE can be further divided into the isolation technics such as capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), capillary isotachophoresis (cITP) and capillary electric chromatogram method (CEC).A kind of embodiment that the CE technology is combined with electro-spray ionization comprises the use volatile solvent soln, for example, contains volatile acid and/or alkali and such as the organic aqueous mixture of ethanol or acetonitrile.

Capillary isotachophoresis (cITP) is a kind of like this technology, and wherein, analyte moves by kapillary with constant speed, but separates according to its movability separately.Capillary zone electrophoresis (CZE), also be free solution C E (FSCE), based on the difference of the electrophoretic mobility of material, the difference of electrophoretic mobility depends on the frictional resistance that electric charge on the molecule and molecule meet with in transition process, and frictional resistance is directly proportional with molecular size usually.Capillary isoelectric focusing (CIEF) allows the amphipathic molecule of faint ionization to separate by the electrophoresis in the pH gradient.CEC is traditional high performance liquid chromatography (HPLC) and the hybrid technology of CE.

Separation that the present invention is used and purification technique comprise arbitrary chromatography operation known in the art.Chromatography can be based on different absorption and the wash-out or the distribution of analyte between moving phase and stationary phase of specific analyte.Chromatographic different example includes but not limited to liquid phase chromatography (LC), vapor-phase chromatography (GC), high performance liquid chromatography (HPLC) etc.

Determine the PARP level

Poly-(ADP-ribose) polymerase (PARP) is also referred to as poly-(ADP-ribose) synzyme and poly-ADP ribosyltransferase.The formation of poly-(ADP-ribose) polymkeric substance of PARP catalysis, this polymkeric substance can be connected on the nucleoprotein (also being connected in himself), thereby has changed these activity of proteins.This kind of enzyme is playing a role aspect the raising DNA reparation, (summary see people such as D.D ' amours " Poly (ADP-ribosylationreactions in the regulation of nuclear functions, " Biochem.J.342:249-268 (1999)) also plays a role aspect the nuclear chromatin regulating.

PARP-1 comprises that the terminal DNA of N-is in conjunction with the territory, modify the terminal catalytic domain of territory (auto modificationdomain) and C-and various cell protein and PARP-1 interaction certainly.The terminal DNA of N-contains two zinc-finger motifs in conjunction with the territory.X acceptor α, the archaeal dna polymerase α of the transcriptional enhancer factor-1 (TEF-1), biostearin, X-ray repair the cross complementary factor-1 (XRCC1) and PARP-1 itself interacts in this territory with PARP-1.Contain the BRCT motif from modifying the territory, it is a kind of protein-protein interaction module.This motif finds in the C-end of BRCA1 (breast cancer sensitive protein 1) at first, and is present in various and DNA reparation, reorganization and the cell cycle check point control proteins associated matter.The eight aggressiveness transcription factors-1 (Oct-1), Yin Yang (YY) 1 and the ubiquitin binding enzyme 9 (ubc9) that contain the POU-homeodomain can interact with this BRCT motif among the PARP-1.

The 15 kinds of members that surpass of PARP gene family are present in the mammalian genes group.PARP family protein and will gather poly-(ADP-ribose) glycosylhydrolase (PARG) that (ADP-ribose) is degraded to ADP-ribose, participation comprises that dna damage replys the various kinds of cell regulatory function with transcriptional regulatory, and relates to the biology of carcinogenesis and cancer in many aspects.

Several PARP family proteins have been identified.Found to hold the interaction protein of anchor polymerase (Tankyrase), and participated in the telomere adjusting as telomere regulatory factor 1 (TRF-1).Fornix (Vault) PARP (VPARP) is a kind of composition in the fornix compound, and the fornix compound plays a role as nuclear-tenuigenin transporter.PARP-2, PARP-3 and 2,3,7 have also been identified, the 8-tetrachloro dibenzo-derivable PARP of right-dioxin (TiPARP).Therefore, poly-(ADP-ribose) metabolism may be relevant with the various kinds of cell regulatory function.

A member of this gene family is PARP-1.PARP-1 gene outcome high level expression in nucleus, and its activation depends on dna damage.Be not subjected to the restriction of any theory, think that PARP-1 passes through amino terminal DNA in conjunction with the fracture of territory in conjunction with dna single chain or two strands.This combination has activated the carboxyl terminal catalytic domain, and causes the formation of polymkeric substance on target molecules of ADP-ribose.PARP-1 itself is the target that relies on the poly-ADP-ribosylation of modifying the territory certainly that is positioned at the center.The ribosylation of PARP-1 causes the PARP-1 molecule to dissociate from DNA.Take place very fast in conjunction with, the ribosylation and the whole process of dissociating.Shown that PARP-1 causes replenishing of DNA repair mechanism with this of short duration combination the in dna damage site, maybe can be used for suppressing sufficiently long reorganization for repair mechanism replenishes.

The source of the ADP-ribose of PARP reaction is a niacinamide adenosine dinucleotide (NAD).By the synthetic NAD of cell ATP deposit, therefore the activation of high-caliber PARP activity can cause exhausting of cellular energy deposit fast in cell.Proved that inducing of PARP activity can cause consuming relevant cell death with cellular NAD and ATP storehouse.Induce the PARP activity in the situation of many oxidative stresss or in the inflammatory process.For example, in the refilling process of ischemic tissue, produce reactive nitrogen monoxide, and nitrogen monoxide causes producing other reactive oxygen species, comprise hydrogen peroxide, peroxynitric acid salt and hydroxyl radical free radical.Back these materials meeting coup injuries DNA, and the activation of PARP activity is induced in the damage that produces.Frequently, as if the abundant activation of PARP activity taking place, makes cellular energy storage depletion and cell death.It is believed that, when endothelial cell and short inflammatory cell are synthetic when causing the nitrogen monoxide of O-DNA damage in the peripheral cell and the active activation of PARP subsequently, the similar mechanism of operation in inflammatory process.It is believed that the cell death that PARP activation causes is the main contribution factor of the degree of tissue damage that causes of ischemia reperfusion damage or inflammation.

The treatment that is suppressed at of PARP activity may be useful in the cancer.The disinthibiting and to break by the specific DNA of priming cancer cell of DNA enzyme (suppressing), and cancer cell specific induction of apoptosis only by PARP-1.The PARP micromolecular inhibitor can make the kill and wound sensitivity of the tumor cell line of treatment for ionising radiation and some dna damage chemotherapeutic drug.The single therapy of PARP inhibitor or with the therapeutic alliance of chemotherapeutant or radiation be effective treatment.Chemotherapeutant this during as invalid concentration, with disappearing of the therapeutic alliance meeting induced tumor of chemotherapeutant.In addition, PARP-1 mutant mice and PARP-1 mutant clone are responsive for the chemotherapeutic drug of radiation and similar type.

An aspect of of the present present invention relates to determines that disease can use the PARP modulators for treatment such as the PARP inhibitor, wherein, and the determining of disease based on the PARP level of determining the experimenter.A kind of preferred embodiment in, if experimenter's PARP to adjusted, uses PARP inhibitor for treating experimenter so.Compare with the normal subjects, suffer from relative level that the patient's of prostate cancer and breast cancer PARP-1 expresses to adjusted.Similarly, compare, suffer from relative level that the patient's of oophoroma and carcinoma of endometrium PARP-1 expresses to adjusted with the normal subjects.In different cancers, every kind of cancer types show the degree that differs from one another to adjusted.For example, different breast cancer showed different to adjusted.Similarly, different oophoroma showed different to adjusted.What show PARP-1 not only helps the disease of definite PARP-1 mediation can use the PARP-1 inhibitor for treating to adjusted, and helps to predict/determine according to the upwards regulating degree of PARP-1 among the experimenter curative effect of PARP-1 inhibitor.The assessment of PARP-1 gene expression can be the indication of tumour to the susceptibility of PARP-1 inhibitor.It also helps according to giving experimenter's personalized design dosage to the PARP-1 of adjusted level.

In some embodiments, the PARP level in patient's sample and the default standard model relatively.Patient's sample is generally from pathological tissues, as cancer cell or cancerous tissue.Standard model can be from same patient or different experimenters.Standard model is generally normal, non-pathology sample.But, in some embodiments, for example for to the disease classification or the assessment result of treatment, standard model is from pathological tissues.Standard model can be the combination from several different experimenters' sample.In some embodiments, compare patient's PARP level and the level that presets.The level that presets generally obtains from normal specimens." the PARP level that presets " as herein described can be the PARP level that is used for following purpose: only for instance, be used to assess the selected patient who treats, be used to assess response for the PARP inhibitor for treating, be used to assess response for the therapeutic alliance of PARP inhibitor and second kind of therapeutic agent, and/or the patient of cancer diagnosis, inflammation, pain and/or relevant disease.The PARP level that presets can suffer from or not cancered patient colony in measure.The PARP level that presets can be the independent numerical value that is equally applicable to each patient, and the PARP level that perhaps presets can change with specific patient subgroups.For example, the male sex has the PARP level that preset different with the women, and the non-smoker has the PARP level that preset different with the smoker.Patient's age, body weight and height can influence the individual PARP level that presets.In addition, the PARP level that presets can be the level that each patient is determined separately.The PARP level that presets can be any suitable standard.For example, can select the same people that assesses or different people to obtain the PARP level that presets from it being carried out the patient.In one embodiment, can be from the PARP level that obtains for the assessment before the same patient to preset.By this way, along with the development of time monitoring patient's selection.In addition, also can obtain standard from another person or a plurality of people's (for example, selected group people) assessment.By this way, select the people's that assesses selection degree to compare with suitable other people to it, for example, situation is similar to other people of interested people, suffers from the people of similar or identical disease as those.

In one embodiment of the invention, PARP is with respect to about 0.5 times of changing into of the level that presets, about 1.0 times, about 1.5 times, about 2.0 times, about 2.5 times, about 3.0 times, about 3.5 times, about 4.0 times, about 4.5 times or about 5.0 times.In some embodiments, change multiple be lower than about 1, be lower than about 5, be lower than about 10, be lower than about 20, be lower than about 30, be lower than about 40 or be lower than about 50.In other embodiments, compare the level that presets, the change of PARP level greater than about 1, greater than about 5, greater than about 10, greater than about 20, greater than about 30, greater than about 40 or greater than about 50.Preferably the change multiple with respect to the level that presets is about 0.5, about 1.0, about 1.5, about 2.0, about 2.5 and about 3.0.

Below shown in Table I-XXIII for example understand PARP-1 gene expression data in the experimenter who suffers from cancer, metabolic disease, endocrine and neuroendocrine obstacle, angiocardiopathy (CVS), central nervous system disease (CNS), disease in the male sexual system, disease in the female sexual system, respiratory disease, urethra obstacle, inflammation, blood-lymphatic system obstacle and digestive system obstacle.The PARP approach comprises negative effect device, opposite effect and the TNFR1 signal pathway of AIF in apoptosis and cell survival of the apoptotic signal approach, the Caspase cascade in apoptosis, D4-GDI signal pathway, FAS signal pathway (CD95), HIV-I Nef:Fas and the TNF that respond dna damage.

In all forms, C is contrast, and E is a laboratory sample, and SD is a standard deviation, and FC is the change multiple of expression.Expression intensity size in Table II is 0,187.0,374.0,561.0 and 748.Expression intensity size in Table IV is 0,206.0,412.0,617.0 and 823.Expression intensity size in Table VI and Table VII is 0,97.0,194.0,291.0 and 388.Expression intensity size in Table X V is 0,139.0,278.0,417.0 and 556.Expression intensity size in Table X VIII is 0,250.0,500.0,750.0 and 999.Expression intensity size in Table X XII is 0,132.0,264.0,397.0 and 528.Expression intensity size in Table X XIII is 0,180.0,360.0 and 541.0.

FC is on the occasion of the PARP-1 of representative to adjusted, and FC is the PARP-1 that the negative value representative is regulated downwards.Therefore, the present invention has determined to have the various diseases to the PARP-1 of adjusted, and it can be by the PARP-1 inhibitor for treating; And the present invention has also determined to have the various diseases of the PARP-1 of downward adjusting, and it can be by PARP-1 activator or agonist treatment.Table I has represented to have the various cancers to the PARP-1 of adjusted, and for example, seedling is reined in mixed tumour (mullerianmixed tumor), wilms' tumor, serous cystadenocarcinoma etc.Table I has also represented to have the cancer of the PARP-1 of downward adjusting, for example, and the adenocarcinoma metastatic of Hashimoto thyroiditis, benign protuberance hyperplasia, gland carcinoma squamosum, islet-cell tumour and stomach etc.Therefore, the present invention has determined to have the various diseases to the PARP-1 of adjusted, and it can be by the PARP-1 inhibitor for treating; And the present invention has also determined to have the various diseases of the PARP-1 of downward adjusting, and it can be by PARP-1 activator or agonist treatment.

Table III shows the PARP-1 to adjusted of various breast cancer, and wherein, the infiltrating carcinoma of hybrid catheter and microphyll type shows the PARP-1 that regulates downwards.Table VIII shows through drug therapy with without the experimenter's of drug therapy PARP-1 level.Table X shows the various respiratory diseases that have to the PARP-1 of adjusted, and wherein, essential gland carcinoma squamosum shows the PARP-1 that regulates downwards.Table X II is presented at the expression of the PARP-1 among the experimenter who contrasts the experimenter and suffer from inflammation, and has illustrated the PARP-1 to adjusted and downward adjusting among the ill experimenter.Table X VI is presented at the expression of the PARP-1 among the experimenter who contrasts the experimenter and suffer from the CNS disease, and has illustrated the PARP-1 to adjusted and downward adjusting among the ill experimenter.Table X IX is presented at the expression of the PARP-1 among the experimenter who contrasts the experimenter and suffer from the blood-lymphatic system obstacle, and has illustrated the PARP-1 to adjusted and downward adjusting among the ill experimenter.Table X XI is presented at the expression of the PARP-1 among the experimenter who contrasts the experimenter and suffer from various endocrines and neuroendocrine system obstacle, and has illustrated the PARP-1 to adjusted and downward adjusting among the ill experimenter.

The invention provides method for supervising, wherein, in the process of cancer or antineoplaston, also preferably, before treatment and during begin treatment, monitoring cancer patient's PARP level.Compare the PARP level of the normal individual that does not have cancer, the reduction of cancer patient's PARP level or the mensuration of increase allow followingly to make progress and/or the relevant assessment of result with the patient: (i) more serious cancer staging or classification; The (ii) shorter progression of disease time; And/or (iii) the patient lacks positive response to treatment of cancer, that is, and and effective response.For example, according to respect to normal PARP level and with respect to the time dependent PARP level of measuring before the patient self of horizontal monitored patient, can determine whether therapeutic scheme should change, that is, therapeutic scheme should be more positive still more conservative; To determine that whether the patient is for the positive response of its treatment; And/or the state of definite disease, the alleviation of for example late period of cancer, or cancer or neoplastic disease, dwindle or disappear.The present invention allows to determine clinical benefit, progress time (TTP) and time-to-live length based on the data of comparing with the normal individual level to adjusted or downward PARP level of regulating.The present invention also comprises the PARP diagnostics and uses the method for these diagnostics.

Analysis to patient's PARP level is especially valuable and useful, because this makes the doctor can more effectively select optimal treatment, and according to the adjusted or the PARP level of regulating, uses more positive therapeutic and therapeutic scheme downwards.The more combination of positive therapeutic or treatment and scheme can be offset patient's relatively poor prognosis and total life span.Utilize this information, the medical personnel can select to provide the treatment of some type, as the PARP inhibitor for treating, and/or positive therapeutic more.

When following period of time monitoring patient's PARP level, (can be a couple of days, several weeks, several months during this period of time, in some cases, it can be the several years, or its various intervals), patient's humoral sample, for example serum or blood plasma can be collected in the interval of being determined by the medical personnel such as doctor or clinician, determine the PARP level, and in process or treatment or disease, compare with the level of normal individual.For example, can be according to the present invention, with every month, per two months or one, two or time interval of combination in three months, gather also monitoring patient sample.In addition, in the monitoring phase, the patient's who change to obtain in time PARP level can be compared to each other easily, and with the PARP value of normal control relatively, thereby provide patient's oneself PARP value, inside or the individual contrast of monitoring as long-term PARP.

The PARP analytical technology

PARP analyzes the analysis comprise PARP gene expression, comprises that DNA, RNA analyze, and the analysis of PARP level, and/or the analysis of PARP activity comprise single-and the level of many-ADP-riboseization.Scope of the present invention without limits can adopt the technology known in the art of arbitrary number to be used for the PARP analysis, and they within the scope of the present invention.Provided some examples of such detection technique below, can be used for various detection technique of the present invention but these examples are in no way limited to g.

Gene expression spectrum analysis: the method for gene expression spectrum analysis comprises the method based on the hybridization analysis of polynucleotide, polyribonucleotide, based on the method for polynucleotide, polyribonucleotide sequencing with based on the method for protein science.The method that mRNA in the most frequently used quantitative sample known in the art expresses comprises Northern blotting and in situ hybridization (Parker ﹠amp; Barnes; Methods in Molecular Biology 106:247-283 (1999)), (Hod is measured in the RNAse protection; Biotechniques 13:852-854 (1992)) with such as the method (people such as Weis, Trends in Genetics 8:263-264 (1992)) of the PCR-based of RT-polymerase chain reaction (RT-PCR).Perhaps, the antibody that to discern specific duplex be can adopt, DNA duplex, RNA duplex and DNA-RNA heteroduplex body or DNA-protein duplex comprised.Comprise serial analysis of gene expression (SAGE) and gene expression analysis, comparative genome hybridization (CGH), chromatin immuno-precipitation (ChIP), single nucleotide polymorphism (SNP) and SNP array, fluorescence in situ hybridization (FISH), protein combination array and dna microarray (being also referred to as gene or genome chip, DNA chip or gene array usually), RNA microarray by extensive parallel signature order-checking (MPSS) based on the exemplary process of the gene expression analysis of sequencing.

Reverse transcriptase PCR (RT-PCR): a kind of the sensitiveest and be RT-PCR based on the gene expression spectrum analysis method of quantitative PCR the most flexibly, its can be used for comparison normal structure and tumor tissues (with or do not heal with medicine) different sample colony in the mRNA level, characterize gene expression pattern, the closely-related mRNA of difference and analyze the RNA structure.

The first step is a separating mRNA from target sample.For example, parent material is generally respectively the total RNA that separates from people's tumour or tumor cell line and corresponding normal structure or clone.Therefore, isolation of RNA can and organizing from multiple normal and ill cell for example, comprises the tumour or the tumor cell line in breast, lung, colorectum, prostate, brain, liver, kidney, pancreas, spleen, thymus gland, testis, ovary, uterus etc.If the source of mRNA is a primary tumo(u)r, can be from (for example) extract mRNA the freezing or fixing tissue preserved, for example the tissue sample of paraffin embedding and fixing (for example, formalin fixed).The conventional method that extracts mRNA is to know in this area, and has in molecular biological standard textbook openly, comprises people such as Ausubel, Current Protocols of Molecular Biology, John Wiley and Sons (1997).

Specifically, can use purification kit, damping fluid group and proteinase to carry out separating of RNA according to the instructions of manufacturer from commercial suppliers.For example, can separate from the RNA of tumour preparation by cesium chloride density gradient centrifugation.Because RNA can not be as the template of PCR, the first step by RT-PCR analyzing gene express spectra is to be cDNA with RNA template reverse transcription, index amplification in the PCR reaction then.Two kinds of the most frequently used reverse transcriptases are AMB (avilo myeloblastosis) viral reverse transcriptase (AMV-RT) and Moroni murine leukemia virus reverse transcriptase (MMLV-RT).Use Auele Specific Primer, hexamer or the initiation of oligo-dT primer at random as the reverse transcription step 1, this depends on the target of environment and expression pattern analysis.The template that the cDNA that derives can react as PCR subsequently then.

For effect wrong and the sample room variation is minimized, mark carries out RT-PCR in using usually.Be marked in desirable between the different tissues and express, and do not tested the influence of processing with constant level.It is the most frequently used that to come the RNA of standardization gene expression pattern be the mRNA of house-keeping gene glyceraldehyde-3-phosphate-dehydrogenasa (GAPDH) and beta-actin.

The newer variation of RT-PCR technology is a real-time quantitative PCR, and it measures the accumulation of PCR product by the product fluorescence probe of double labeling.PCR in real time and quantitative competitive PCR and quantitative comparison PCR are compatible, in quantitative competitive PCR, are used for standardization for the internal competition agent of each target sequence; Quantitative comparison PCR uses standardization gene that comprises in the sample or the house-keeping gene that is used for RT-PCR.

Fluorescence microscopy: some embodiment of the present invention comprises the fluorescence microscopy that is used for the PARP analysis.Fluorescence microscopy makes it possible to by using the fluorescently-labeled molecular composition of determining observed structure such as the height chemistry specific probe of antibody.Can be by with fluorophore and the direct coupling of protein and be introduced into back in the cell and carry out.The fluorescence analog can have the behavior as native protein, thereby can be used for showing distribution and the behavior of this protein in cell.Together with NMR, infrared spectrum, circular dichroism and other technology, the observation of the fluorescence decay of protein inherence and the fluorescence anisotropy of combination, collisional quenching and resonance energy transfer is the protein detection technology.Natural fluoresence protein can be used as fluorescence probe.Jellyfish (jellyfish aequoreavictoria) produces a kind of natural fluoresence protein that is called green fluorescent protein (GFP).The fusion of these fluorescence probes and target protein allows quantitatively to show by fluorescence microscopy and flow cytometry.

Below only be for example, some probe is following label, replaces thing, long-life, long wavelength's the label (800nm at most) and compound, DY flower cyanines label and the phycobniliprotein (phycobili proteins) of lanthanide series as fluorescein and derivant, Fluoresceincarboxylic acid, rhodamine and derivant thereof, atto label, fluorescein and fluorescent orange: cy3/cy5.Below only be that some probe is following conjugate, such as isothiocyanates conjugate, streptavidin conjugate and biotin conjugates for example.Below only be that some probe is following zymolyte, as fluoresce substrate and chromogenic substrate for example.Below only be for example, some probe is following fluorescent dye, as FITC (green fluorescence, excitation/emission=506/529nm), rhodamine B (fluorescent orange, excitation/emission=560/584nm) and Nile blue A (red fluorescence, excitation/emission=636/686nm).Fluorescent nano particles can be used for various types of immunoassays.Fluorescent nano particles is based on different materials, as polyacrylonitrile and polystyrene etc.The fluorescence molecule rotor is the sensor of microenvironment restriction, when it rotates when limited, becomes epipolic.Several examples of molecule constraint comprise the dyestuff (gathering), binding antibody of increase or are caught by the polymerization of actin.IEF (isoelectric focusing) be used to separate mainly be protein ampholyte analysis tool.An advantage with IEF gel electrophoresis of fluorescence IEF label be can the Direct observation gradient formation.Also can absorb (20 ℃) by the UV of 280nm and detect fluorescence IEF label.

Can be on solid support by using colorant receptor synthesize peptide library, the solid support of selective staining one by one subsequently.If acceptor can not show any color, their binding antibody can be colored.This method not only can be used for protein acceptor, also can be used to screen the binding partner of synthetic artificial receptors and screen new metal binding partner.Also can use the automatic mode that is used for HTS and FACS (fluorescent activation cell sorter).The FACS instrument moves cell by kapillary when beginning, and by detecting its fluorescence intensity isolated cell.

Immunoassays: some embodiment of the present invention comprises the immunoassays that are used for the PARP analysis.In the Western blot as the immunoblotting of electrophoretic separation protein, a kind of protein is determined by its antibody.Immunoassays can be that the competition binding immunoassay is measured, wherein, the limited amount antibody molecule of the antigenic competition of analyte and mark (for example, radioimmunoassay, EMIT).Immunoassays can be noncompetitive, and wherein, antibody excess exists, and is labeled.Along with the increase of analyte antigen compound, the amount of the antibody-antigenic compound of mark also can increase (for example, ELISA).If produce by antigen is injected into animal used as test, antibody can be polyclonal; Perhaps, if produced by Fusion of Cells and cell culture technology, antibody can be monoclonal.In immunoassays, antibody can be used as the specific reagent of analyte antigen.

Scope of the present invention without limits and content, below only be for example, some type of immunoassays is: RIA (radiommunoassay), as enzyme immunoassay (EIA), LIA (luminescent immunoassay) and the FIA (fluorescence immunoassay) of ELISA (enzyme linked immunological absorbing agent mensuration), EMIT (EMIT), particulate enzyme immunoassay (EIA) (MEIA).These technology can be used for detecting the biological substance in the nose sample.Antibody---no matter be to use as first antibody or second antibody, can use radioactive isotope (for example, 125I), fluorescent dye (for example, FITC) can catalysis product fluorescence or the enzyme (for example, HRP or AP) of luminescence-producing reaction come mark.

Biotin (or biotin) is a kind of coenzyme, and it has the specificity affinity for avidin and streptavidin.This interaction makes biotinylated peptide become a kind of useful instrument that the various biotechnologys that are used for qualitative and detection by quantitative are measured.In order to improve the identification of biotin/streptavidin by the minimize spatial steric hindrance, must enlarge the distance of biotin and peptide itself.This can realize by connect spacer molecule (for example, 6-aminocaprolc acid) between biotin and peptide.

Biotin quantitative measurement for biotinylated protein provides a kind of fluorometric assay that is used for accurately measuring the sensitivity of the biotin labeling quantity on the protein.Biotinylated peptide is widely used in the multiple biomedical screening system, and these biomedical screening systems need be fixed at least a interaction gametophyte on pearl, film, microslide or the microtiter plate of streptavidin bag quilt.This mensuration is based on from the part of the biotin binding site displacement quencher dye marker of reagent.For any biotin group in limited and the protein inaccessible multiple labelling of reagent on the exposed space, can be with the Protease Treatment protein of digesting protein.

EMIT is that the competitive binding immunoassay of the separating step of avoiding common is measured.One type immunoassays, wherein protein enzyme labeling, and enzyme-protein-antibody complex does not have enzymatic activity, allows there not being the quantification of protein of mark.Some embodiment of the present invention comprises the ELISA that analyzes PARP.ELISA is based on the antibodies selective that is connected on the solid support, and desmoenzyme reacts, and generation can detect the system of low-level protein.It also is called enzyme immunoassay (EIA) or EIA.Protein its antibody test that has prepared, that is, this protein is antigen.Often use monoclonal antibody.

Detection may need to be fixed on such as the antibody on the solid surface on surface in vitro and with the preparation of the same antibody of enzyme coupling.Enzyme can be the enzyme (for example, beta galactosidase) that produces coloured product from colourless substrate.For example, can detect by filling test tube with antigenic solution to be determined (for example, protein).The antigen molecule of any existence can be in conjunction with fixing antibody molecule.Antibody-enzyme conjugates can be added in the reaction mixture.The antibody moiety of this conjugate produces antibody-Ag-Ab " interlayer " in conjunction with any antigen molecule of combination in the past.After any unconjugated conjugate of flush away, can add substrate solution.Behind the interval of a setting, cessation reaction (for example), and the concentration of the coloured product that forms with spectrophotometric determination by adding 1N NaOH.The intensity of color is proportional with the concentration of the antigen that combines.

ELISA also is applicable to the concentration of measuring antibody, in this case, and with suitable antigen coated hole.Can add the solution (for example, serum) that contains antibody.After the antigen that its free combination is fixed, can add anti-immunoglobulin by the enzyme coupling of forming at the antibody of antibody to be measured.After the unreacted reagent of flush away, add substrate.The intensity proportional with the amount of the antibody of the enzyme labeling that combines (therefore, proportional) of the color that produces with the concentration of the antibody of being tested.

Some embodiment of the present invention comprises the radiommunoassay of analyzing PARP.Radioactive isotope can be used for studying internal metabolism, distribution and the combination of a small amount of compound.In vivo, use ¹H, ¹²C, ³¹P, ³²S and ¹²⁷The radioactive isotope of I, as ³H, ¹⁴C, ³²P, ³⁵S and ¹²⁵I.In the acceptor fixation of 96 orifice plates, by using antibody or chemical method sessile receptor in every hole, and can in every hole, add radiolabeled part and induce combination.Can the unconjugated part of flush away, the radioactivity determination reference material of the part of radioactivity by the quantitative test binding partner or flush away then.Then, the adding of screening target compound can be induced the competitive association reaction with acceptor.If compound shows higher affinity to acceptor than standard radioligand, then most of radioligand will be not can bind receptor and stay in the solution.Therefore, the amount of the radioligand (or part of flush away) by analyzing combination can show the affinity of test compounds to acceptor.

When acceptor can not be fixed in 96 orifice plates, or when part in conjunction with need when solution carries out in mutually, needing filter membrane method.In other words, after the ligand-receptor association reaction in solution, if, comprise that the micromolecule of part can pass through, and have only protein acceptor to stay on the filter paper by cellulose nitrate filter paper filtering reaction solution.Have only consumingly the part of bind receptor to stay on the filter paper, and can determine the relative affinity of the compound that adds by quantitative test standard radioligand.

Some embodiment of the present invention comprises the fluorescence immunoassay of analyzing PARP.Be based on of the competitiveness combination of the part of mark on the acceptor site of high degree of specificity with respect to unlabelled part based on the immunological method of fluorescence.Can use fluorescent technique to carry out immunoassays, it is based on the variation of the fluorescence lifetime that changes with analyte concentration.This technology can be used the short-life dyestuff as fluorescein isothiocynate (FITC) (donor), and the fluorescence of FITC can be transferred to eosin (acceptor) and quencher by energy.Can use many photoluminescent compounds, as flower Jing, oxazine, thiazine, porphyrin, phthalocyanine, the ultrared polynuclear armatic hydrocarbon of emitting fluorescence, phycobniliprotein, side's sour inner salt (squaraines) and organism-metal composite, hydro carbons and azo dyes.

Immunological method based on fluorescence can be (for example) heterogeneous or homogeneity.Heterogeneous immunoassays comprise the physical separation of analyte and the analyte of free mark of the mark of combination.Analyte or antibody can be attached on the solid surface.This technology can be competitive (for higher selectivity) or noncompetitive (for higher susceptibility).Detection can be direct (only using one type antibody) or indirect (using second type antibody).The homogeneity immunoassays do not comprise physical separation.The antigen of double antibody fluorophore mark is participated in and balanced reaction at the antibody of antigen and fluorophore.Mark and unlabelled antigen can be competed with the anti--antigen-antibody of limited quantity.

Some fluorescence immunoassay method comprises simple fluorescence labeling method, fluorescent energy resonance transfer (FRET), time-resolved fluorescence (TRF) and Scanning Probe Microscopy (SPM).Simple fluorescence labeling method can be used for the receptor-ligand combination, pass through to use the enzymatic activity of relevant fluorescence and the fluorescence indicator that changes as the various body physiologicals such as pH, ion concentration and voltage.TRF is a kind of after other fluorescence molecule emission finishes, and optionally measures the method for the fluorescence of lanthanide series.TRF can use with FRET, and lanthanide series can become donor or acceptor.In Scanning Probe Microscopy, in catching mutually, at least a monoclonal antibody is attached on the solid phase, and the antigen/antibody compound that utilizes scanning probe microscopy to detect may to be present on the solid phase surface.The needs to mark have been eliminated in the use of scanning tunnel microscope, and this mark is used to detect the antigen/antibody compound usually in many immunoassay systems.

The identification of proteins method: following only is for example, the identification of proteins method comprise by the small throughput sequencing of Edman degraded, mass-spectrometric technique, peptide quality fingerprinting analyze, sequencing and from the beginning based on the analysis of antibody.Quantification of protein measure comprise that fluorescent dye is gel-colored, mark or chemical modification method (that is, isotope-coded affinity label (ICATS)), combined classification diagonal chromatography (combined fractional diagonal chromato graphy) (COFRADIC).The protein of purifying also can be used for the mensuration of three-dimensional crystalline structure, and it can be used for the interaction between model molecule.The commonsense method of measuring three-dimensional crystalline structure comprises X-radiocrystallography and NMR spectroscopy.Can survey the feature of indicator protein matter three-dimensional structure with mass spectroscopy.By use chemical crosslinking come on the coupling space near but the part of a good distance away protein on the sequence can infer about integrally-built information.By the exchange of the deuterium in amide proton and the solvent, can survey the solvent accessibility of the various piece of protein.

In one embodiment, use fluorescent activation cell sorter (FACS) to determine the PARP express cell.FACS is a kind of flow cytometer of specialized types.It provides a kind of special light scattering and fluorescent characteristics according to each cell, and method in two or more containers is assigned to the heterogeneous mixture of biological cell in next cell ground.It provides the quantitative record of the fluorescence signal that individual cells sends and the physical separation of interested cell.In another embodiment, use the expression of assessing PARP based on the equipment of microfluid.

Also can use the PARP of mass spectroscopy sign from patient's sample.Two kinds is electrospray ionization (ESI) and substance assistant laser desorpted/ionization (MALDI) with the Ionized method of whole protein.At first, complete protein is introduced in the mass analyzer then by any ionization in above-mentioned two kinds of technology.Secondly, using such as trypsase or pepsic reagent is less peptide with protein digestibility.Also can use other proteolytic digestion agent.Then the peptide prod of collecting is introduced in the mass analyzer.This is commonly called " (bottom-up) from bottom to top " protein analysis method.

Use flight time (TOF) mass spectrum or Fourier transform ion cyclotron resonance (FT-ICR) to carry out the mass spectrophotometry of complete protein.The instrument that is used for the peptide mass spectrophotometry is a quadrupole ion trap.Multistage four utmost points-flight time and MALDI flight time instrument also are used for this application.

Be used for two kinds of methods with the peptide prod fractionated of protein or its enzymic digestion.First method is separated complete protein fractionation, is called two-dimensional gel electrophoresis.Second method is a high performance liquid chromatography, is used for after enzymic digestion the peptide fractionated.In some cases, must be in conjunction with these two kinds of technology.

Two kinds of methods are arranged, can use the mass spectroscopy identification of protein.The peptide mass spectrum uses the input of the quality of proteoclastic peptide as the database retrieval of predicted quality, and this database source is from the digestion of known protein tabulation.If the protein in the reference listing that produced meaningful quantity with the forecast quality number experiment value coupling, so just have some in primary sample, to have the evidence of this protein.

Tandem mass spectrometry also is a kind of method of identification of protein.Dissociating of collision-induced is used for producing one group of fragment in mainstream applications from the particular peptide ion.Shattering process mainly produces along the dissociating product of peptide bond rupture.

Described many different algorithms, identified peptide and protein by the from the beginning sequencing of tandem mass spectrometry (MS/MS), peptide with based on the retrieval of sequence label.A kind of selection of having made up the data analysis feature of full range is PEAKS.Other existing mass spectrophotometry software comprises: fragments of peptides fingerprint analysis SEQUEST (Mascot, OMSSA and Tandem).

Also can pass through the mass spectroscopy quantitative protein.Usually, stable (for example, inactive) heavy isotope of carbon (C13) or nitrogen (N15) is joined in the sample, another sample is then used corresponding light isotope (for example, C12 and N14) mark.Before analysis, mix these two kinds of samples.Can distinguish peptide according to the difference of its quality from different samples.The ratio of its peak intensity is corresponding to the relative abundance ratio of peptide (and protein).Isotope-labeled method has SILAC (with the amino acid in the stable isotope labeling cell culture), the enzymatic O18 mark of tryptose, ICAT (isotope-coded affinity mark), ITRAQ (being used for relatively and the isotope labeling of absolute quantitationization).Also mark sample not carries out " sxemiquantitative " mass spectroscopy.Usually, carry out this mass spectrum with maldi analysis (linear model).The peak intensity of individual molecule herein (being generally protein) or peak area are relevant with protein content in the sample.But single signal depends on the complicacy of the primary structure of protein, sample and the setting of instrument.

The N-end sequence is measured the evaluation that helps agnoprotein matter, confirm the identity and the degree of accuracy (reading frame, translation starting point etc.) of recombinant protein, help the explanation of NMR and crystallographic data, identical degree between the proof protein, or for the design of the synthetic peptide that produces antibody provides data, or the like.The N-end sequence is measured and is utilized Edman degraded chemistry, removes amino acid residue continuously from the N-end of protein, and determines them by reversed-phase HPLC.Sensitivity can reach the level that 100s flies mole, and often can obtain long sequence reading (20-40 residue) from the starting material of a spot of 10s picomole.Pure protein (〉 90%) can produce the data of easy explanation, but the protein mixture of insufficient purifying also can provide the useful data of carrying out strict data interpretation.End modified (especially acetylizad) protein of N-can not directly check order, and has stoped the Edman chemistry because it lacks free one-level amino.But the limited proteolysis of the protein of blocking-up (for example, using cyanogen bromide) can allow to produce ispol in each circulation of device, and in order to explain significant sequence information, it can carry out data analysis.It is posttranslational modification that the C-end sequence is measured, and influences the structure and the activity of protein.The various diseases situation may be relevant with impaired protein processing, and the C-end sequence is provided by the extra instrument that is used to study protein structure and treatment mechanism that provides.

Determine that disease can use the PARP inhibitor for treating

Some embodiment of the present invention relates to definite a kind of disease can use the PARP modulators for treatment, comprise the PARP level in the sample of determining the experimenter, make the decision that can use the PARP modulators for treatment about definite this disease, wherein, this decision is to make according to the level of PARP.Determine that the PARP level comprises the analysis of RNA, the analysis of PARP level and/or the analysis of PARP activity.When the PARP level in the disease be to adjusted the time, can use this disease of PARP inhibitor for treating.In some embodiments, the PARP level is used for determining the disease relevant with vascularization.

In one embodiment, PARP is to the embodiment of adjusted as the cancer of BRCA defective, and PARP can be used for determining that to adjusted the BRCA cancers mediated can use the PARP modulators for treatment.In another embodiment, determine the mark that adjusting that the PARP level can be repaired as the DNA that the two strands by homologous recombination (HR) is destroyed changes, and the PARP level can be used for making about determining that a kind of disease can be with the decision of PARP modulators for treatment.Determine that the PARP level can comprise the comparison of one or more and reference sample.Can obtain reference sample from following source: identical experimenter, or different experimenters, they or be not subjected to the influence (as, normal subjects) of disease or be the patient.Reference sample can obtain or synthetic the generation from an experimenter, a plurality of experimenter.Determine also can comprise the comparison of appraising datum and database.One embodiment of the present invention relate to the experimenter's who determines to suffer from disease PARP level, and it is associated with normal subjects's PARP level.In some embodiments, carry out the step of related PARP level by software algorithm.Preferably, be computer-reader form with the transformation of data that produces; And, carry out data based user's input parameter is carried out classification algorithms in order to detect the signal of the PARP level of representing ill patient and normal subjects.

Definite and the analysis of PARP level has many treatments and diagnostic application.Clinical practice comprises, for example, the detection of disease, difference morbid state with inform prognosis, such as select with the treatment of PARP inhibitor for treating and/or the prediction of the monitoring of the prediction of the determining of the prediction of treatment response, staging, lysis, result of treatment, patient disease development track (for example, before seizure of disease), bad reaction, with treat relevant effect and the monitoring of toxicity and the detection of recurrence.

Experimenter's the disease of determining and determine subsequently of PARP level disclosed by the invention can be used the PARP inhibitor for treating, can be used for allowing or helping the drug discovery process of therapeutic agent.The definite of PARP level can be used for to adding the patient diagnosis disease of clinical testing.The state of determining to indicate the disease of patient of in clinical testing, receiving treatment of PARP level, and be presented at the variation of state in the therapeutic process.Definite effect that can show of PARP level with the PARP inhibitor for treating, and can for the reaction of various treatments the patient be classified according to them.

Method described herein can be used for determining the state of disease of patient.In one embodiment, this method is used for detecting the earliest stages of disease.In other embodiment, this method is used for to the disease classification of determining.In some embodiments, patient, such as doctor or nurse's health care supplier or health care administration person uses experimenter's PARP level to make diagnosis, prognosis and/or select the treatment option, for example uses the PARP inhibitor for treating.

In other embodiment, method described herein can be used for predicting arbitrary individuality for the possibility of the response of particular treatment (as the PARP inhibitor for treating), select treatment or anticipate the possible bad reaction of treatment for particular individual.In addition, this method can be used for assessing treatment effect in time.For example, when the patient receives treatment, can in one period, obtain biological sample from the patient.The PARP level of more different samples is determined the effect of this treatment mutually.In addition, method described herein can be used for the response for one or more treatments of the effect of more different disease treatments and/or different colony (for example, race, family history etc.).

Some preferred embodiment in, use as shown in Figure 2 computing machine to carry out at least one step of method of the present invention.Fig. 2 shows the computing machine of the operation be used to implement the selection relevant with the inventive method.Computing machine 200 comprises the central processing unit 201 that is connected with one group of input/output device 202 by system bus 203.Input/output device 202 can comprise keyboard, mouse, scanner, FPDP, video monitor, LCD, printer etc.Take the storer 204 of primary memory and/or supplementary storage form also to be connected with system bus 203.These assemblies of Fig. 2 have been represented a station symbol semicomputer.This standard computer is programmed according to the present invention.Especially, computing machine 200 can be programmed and be carried out the various operations of method of the present invention.

The storer 204 of computing machine 200 can be stored identification module 205.In other words, identification module 205 can carry out and the step 102 of Fig. 1,103 and 104 relevant operations.Term used herein " identification module " comprises, but be not limited to: the PARP in analysis experimenter's the sample, randomly the PARP horizontal data of compare test sample and reference sample is determined the PARP in the sample, determine disease, and further definite this disease can be used the PARP inhibitor for treating.Identification module also can comprise decision module, wherein, decision module comprises executable instruction, makes about determining that this disease can be with the decision of PARP inhibitor for treating, and/or provides conclusion about disease to patient, health care supplier or health care administration person.The executable code of identification module 205 can be used many digital technologies and compare and diagnose.

Some embodiment of the present invention comprises computer-readable medium, it comprises the information that can use the PARP modulators for treatment about experimenter's disease, this information obtains by following steps: determine the PARP level in experimenter's the sample, and make about the decision with PARP modulators for treatment disease according to this PARP level.This medium can comprise the reference model of one or more PARP levels in the sample.This reference model can be used for relatively the pattern that obtains from test subject, and can be according to this analysis of relatively making disease.This reference model can be from normal subjects (that is the experimenter who, does not have disease), have the experimenter of various disease level, the experimenter with disease of different seriousness.These reference models can be used to diagnose, the seriousness of prognosis, assessment treatment validity and/or definite experimenter's morbid state.Method of the present invention is also included between one or more computing machine, and internet usage for example transmits about the information of the PARP level in experimenter's the sample and/or about determining that this disease can be with the decision of PARP inhibitor for treating of the present invention.

Disease

Various diseases comprises, but be not limited to: cancer types comprises adrenocortical carcinoma, cancer of anus, alpastic anemia, cholangiocarcinoma, carcinoma of urinary bladder, osteocarcinoma, bone shifts, adult CNS brain tumor, children CNS brain tumor, breast cancer, the Castleman disease, cervix cancer, children's non_hodgkin lymphoma, the colon and the carcinoma of the rectum, carcinoma of endometrium, the cancer of the esophagus, especially because of family tumor, cancer eye, carcinoma of gallbladder, the stomach and intestine carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, Hodgkin's disease, Kaposi sarcoma, kidney, throat and hypopharyngeal cancer, acute lymphatic leukemia, acute myelogenous leukemia, leukemia of children, chronic lymphocytic leukemia, chronic myelogenous leukemia, liver cancer, lung cancer, the lung carcinoid tumor, non_hodgkin lymphoma, male breast carcinoma, malignant mesothelioma, Huppert's disease, myelodysplastic syndrome, nasal cavity and nasal side cancer, nasopharyngeal carcinoma, neuroblastoma, oral cavity and oropharynx cancer, osteosarcoma, oophoroma, cancer of pancreas, carcinoma of penis, hypophysoma, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary-gland carcinoma, sarcoma (the soft tissue cancer of growing up), the melanoma skin cancer, non-melanoma skin cancer, cancer of the stomach, carcinoma of testis, thymic carcinoma, thyroid cancer, sarcoma of uterus, carcinoma of vagina, carcinoma of vulva, macroglobulinemia Waldenstron (Waldenstrom ' s macroglobulinemia), chronic lymphocytic leukemia and reactive lymphoid hyperplasia.

Disease is included in cancer, inflammation, degenerative disease, CNS disease, autoimmune disease and comprises vascularization in the virus disease of HIV.Compound as herein described also is useful in regulating the cell response of pathogen.The present invention also provides the method for other disease of treatment such as virus disease.Some virus disease includes, but are not limited to: human immunodeficiency virus (HIV), 1 type and herpes simplex types 2 virus and cytomegalovirus (CMV) (the coinfection virus of a kind of danger of HIV).

This paper has proposed some example of disease, but does not limit the scope of the invention, and also has other disease known in the art within the scope of the present invention.

The example of cancer

The example of cancer includes, but are not limited to: the tumour of lymthoma, cancer and dependence hormone (for example, breast cancer, prostate cancer or oophoroma).Treatable abnormal cell hyperplasia situation or cancer comprise entity tumor/malignant tumour among adult or the children, local late tumor, people soft tissue sarcoma, the metastatic cancer that comprises Lymph Node Metastasis, comprise Huppert's disease, acute and chronic leukemia and lymphadenomatous blood cell malignant tumour, comprise a mouthful cancer, the head and neck cancer of laryngocarcinoma and thyroid cancer, the lung cancer that comprises small cell carcinoma and non-small cell carcinoma, the breast cancer that comprises small cell carcinoma and duct carcinoma, comprise cancer of the esophagus, cancer of the stomach, colon cancer, the human primary gastrointestinal cancers of colorectal cancer and the polyp relevant with colorectal carcinoma, cancer of pancreas, liver cancer, the urinary tract cancer that comprises carcinoma of urinary bladder and prostate cancer, comprise oophoroma, the female genital tract malignant tumour of the solid tumor in uterus (comprising endometrium) cancer and the ovary follicle, the kidney that comprises clear-cell carcinoma, comprise the endogenous brain tumour, neuroblastoma, the astrocyte brain tumor, glioma, metastatic cancer cell is invaded the cancer of the brain of central nervous system, the osteocarcinoma that comprises osteoma, the cutaneum carcinoma that comprises chromoma, the tumor development of human skin keratinocyte, squamous cell carcinoma, basal-cell carcinoma, hemangiopericytoma and Kaposi sarcoma.

Of the present invention some preferred embodiment in, cancer comprises: adenocarcinoma of colon, adenocarcinoma of esophagus, hepatocellular carcinoma, squamous cell carcinoma, pancreas adenocarcinoma, islet-cell tumour, rectal adenocarcinoma, GISTs, sdenocarcinoma of stomach, adrenocortical carcinoma, follicular carcinoma, papillary carcinoma, breast cancer, duct carcinoma, lobular carcinoma, intraductal carcinoma, mucous carcinoma, phyllodes tumor, adenocarcinoma ovaries, adenocarcinoma of endometrium, GCT, MCAC, adenocarcinoma of cervix, the vulva squamous cell carcinoma, basal-cell carcinoma, adenocarcinoma of the prostate, giant cell tumor of bone, the bone osteosarcoma, laryngocarcinoma, adenocarcinoma of lung, kidney, carcinoma of urinary bladder and wilms' tumor.

Of the present invention further preferred embodiment in, cancer comprises: endometrial seedling is reined in mixed tumour, conduit and leaflet mixed type infiltrating cancer, wilms' tumor, the seedling of ovary is reined in mixed tumour, serous cystadenocarcinoma, adenocarcinoma ovaries (palilate slurry type), adenocarcinoma ovaries (endometrial-like type), metastatic wellability lobular carcinoma of breast, seminoma of testis, prostate benign protuberance hyperplasia, squamous cell lung carcinoma, the lung large cell carcinoma, adenocarcinoma of lung, adenocarcinoma of endometrium (endometrial-like type), infitrating ductal carcinoma, rodent ulcer, the mammary gland infiltration lobular carcinoma, the fiber cystic disease, adenofibroma, glioma (gleoma), chronic myelogenous leukemia, hepatocellular carcinoma, mucous carcinoma, neurinoma, the kidney transitional cell carcinoma, Hashimoto thyroiditis, metastatic wellability breast ductal cancer, adenocarcinoma of esophagus, thymoma, phyllodes tumor, rectal adenocarcinoma, osteosarcoma, adenocarcinoma of colon, thyroid papillary carcinoma, liomyoma and sdenocarcinoma of stomach.

Infitrating ductal carcinoma:

Compare with normal level, the expression of the PARP1 in the infiltration ductal carcinomas of breast (IDC) raises.In surpassing 2/3 IDC case, the expression of PARP1 is higher than 95% confidence upper limit (" overexpression ") of normal population.The overexpression of PARP1 takes place in the negative subgroup of the estrogen receptor of IDC (ER) feminine gender and Her2-neu-in about 90% tumour.

An aspect of of the present present invention is with PARP inhibitor for treating IDC.In one embodiment, before using the PARP inhibitor, the state of the expression of PARP and ER and/or progesterone receptor (PR) and/or Her2-neu raises.Preferably, use estrogen receptor negative and the negative subgroup of Her2-neu-of PARP inhibitor for treating IDC.Even more preferably, can not be with the cancer of antiestrogenic or anti-Her2-neu-treatment with the PARP inhibitor for treating.A kind of preferred embodiment in, with the breast cancer of PARP inhibitor for treating triple feminine genders, as the infitrating ductal carcinoma of triple feminine genders.

The cancer of triple feminine genders:

In one embodiment, use the cancer of the triple feminine genders of PARP inhibitor for treating.Preferably, assess PARP level in the cancer of triple feminine genders; And if observed the overexpression of PARP, just with this cancer of PARP inhibitor for treating." triple feminine genders " breast cancer means that this tumour lacks the acceptor for estrogen (ER-feminine gender) and progestational hormone (PR-feminine gender) and HER2 protein.This makes them for some powerful anticancer drug resistances as Tamoxifen, aromatase inhibitor and Trastuzumab (Herceptin).Operation and chemotherapy are the standard care options for triple negative cancers of most of forms.A kind of preferred embodiment in, will combine with the PARP inhibitor for the processing standard of triple negative cancers and treat these cancers.

The example of inflammation:

The example of inflammation includes, but are not limited to: systemic inflammatory situation and with the migration of monocyte, leucocyte and/or neutrophil cell with attract the situation of local correlation.Infection, graft rejection that inflammation can be derived from Pathogenic organisms (comprise gram-positive bacterium, gramnegative bacterium, virus, fungi and such as the parasite of protozoan and worm) (comprise the repulsion such as the solid organ of kidney, liver, the heart, lung or cornea, or be derived from local chronic or acute autoimmunity or allergic reaction and the marrow graft rejection that comprises graft versus host disease (GVHD)).Autoimmune disease comprises acute glomerulonephritis, rheumatoid arthritis or adjuvant arthritis, chronic glomerulonephritis, such as the inflammatory bowel disease of regional enteritis, ulcerative colitis and NEC, syndrome that the granulocyte blood transfusion is relevant, such as the diabetes of contact dermatitis, atopic dermatitis, psoriasic inflammatory dermatosis, systemic loupus erythematosus (SLE), autoimmunity thyroiditis, multiple sclerosis and some form, or the attack of patient's autoimmunity system causes any other autoimmune state of pathologic disorganization.Allergic reaction comprises allergic asthma, chronic bronchitis, acute and delayed allergy.Whole body inflammatory disease state comprise with wound, burn, ischaemic incident (for example, thrombosis incident in the heart, brain, intestines or peripheral vasculature comprises miocardial infarction and apoplexy) afterwards the relevant inflammation of perfusion again, pyemia, ARDS or multiple organ dysfunction syndrome.Raising also of inflammatory cell takes place in atherosclerotic plaque.

Some preferred embodiment in, inflammation comprises: non_hodgkin lymphoma, Wegner's granulomatosis, Hashimoto thyroiditis, hepatocellular carcinoma, atrophy of thymus gland, chronic pancreatitis, rheumatoid arthritis, reactive lymphoid hyperplasia, osteoarthritis, ulcerative colitis, papillary carcinoma, regional enteritis, ulcerative colitis, acute cholecystitis, chronic cholecystitis, sclerosis, chronic sialadnitis, peritonitis, acute pancreatitis, chronic pancreatitis, chronic gastritis, endometriosis interna, endometriosis, acute cervicitis, chronic cervicitis, lymphoid hyperplasia, multiple sclerosis, be secondary to the hypertrophy of ITP, Primary IgA nephropathy, systemic loupus erythematosus, psoriasis, pulmonary emphysema, chronic pyelonephritis and chronic cystitis.

The example of endocrine and neuroendocrine obstacle

Dyshormonal example comprises adrenal gland, mammary gland, sexual gland, pancreas, parathyroid gland, hypophysis, thyroid obstacle, nanism etc.Adrenal obstacle includes, but are not limited to: Addision's disease (Addison ' s disease), hirsutism (hirutism), cancer, multiple endocrine knurl, adrenal,congenital hyperplasia and pheochromocytoma.The obstacle of mammary gland includes, but are not limited to: breast cancer, FBD and gynecomastia.The obstacle of sexual gland includes, but are not limited to: adrenal,congenital hyperplasia, Stein-Leventhal syndrome and Turner syndrome.The obstacle of pancreas includes, but are not limited to: diabetes (I type and II type), hypoglycemia and insulin resistance.Parathyroid obstacle includes, but are not limited to: hyperparathyroidism and hypoparathyroidism.The obstacle of hypophysis includes, but are not limited to: acromegalia, Cushing syndrome (Cushing ' s syndrome), diabetes insipidus, empty sella syndrome, hypopituitarism and prolactinoma.Thyroid obstacle includes, but are not limited to: cancer, goitre, hyperthyroidism, hypothyroidism, tubercle, thyroiditis and Wilson syndrome.The example of neuroendocrine obstacle includes, but are not limited to: depression relevant with hormonal imbalance and anxiety illness, menstrual epilepsy (catamenial epilepsy), menopause, menstruation antimigraine, reproductive endocrine obstacle, such as gastrointestinal tract disorder, achalasia and the Hirschsprung disease (Hirschsprung ' s disease) of the intestines endocrine tumors that comprises carcinoid, gastrinoma and somatostatinoma.In some embodiments, the example of endocrine and neuroendocrine obstacle comprises nodular hyperplasia, Hashimoto thyroiditis, islet-cell tumour and papillary carcinoma.

The example of children's endocrine and neuroendocrine obstacle comprises the endocrinology situation of growth disorder and diabetes insipidus.As observed in holoprosencephaly, septum pellucidum-hypoplasia of optic nerve and the bulging of base portion brain, in having congenital abnormal position or pituitary gland alplasia/undergrown patient, can observe growth delay.Such as craniopharyngioma, eye/acquired situations such as hypothalamus glioma, clinical short stature and diencephalic syndrome can appear.Under following situation, can see sex premature and undue growth: arachnoid cyst, hydrocephalus, hypothalamic hamartoma and gonioma.Pituitary adenoma too much secretes growth hormone and corticotropin can cause pathologic hypomegasoma of children and central obesity.Diabetes insipidus can be secondary to such as behind langerhans cell histiocytosis, tuberculosis, gonioma, the hypophyseal stalk wound/surgical injury and the wellability process of hypoxie-ischemic encephalopathy.

The example of nutrition and metabolism obstacle

The example of nutrition and metabolism obstacle includes, but are not limited to: aspartyl glucose aminuria disease (Aspartylglusomarinuria); biotinidase deficiency; carbohydrates lacks glycoprotein syndrome (CDGS); Ke-Nai syndrome (Crigler-Najjar syndrome); cystinosis; diabetes insipidus; Fabry disease (fabry); the fatty acid metabolism obstacle; galactosemia; Gaucher disease (gaucher); glucose-6-phosphate dehydrogenase (G6PD) (G6PD) disease; glutaric aciduria (glutaricaciduria); hurler syndrome (hurler); Hu Erle-Sha Yi syndrome (hurler-scheie); HH (hunter); hypophosphatemia; the I-cytopathy; krabbe's disease (Krabbe); lactic acidosis; long-chain 3 hydroxyl ethylene reductases lack (LCHAD); lysosomal storage disease; mannosidosis; maple syrup urine disease; Ma Luotuo-Lame syndrome (maroteaux-lamy); metachromatic leukoencephalopathy; mitochondriopathy; morquio's disease (morquio); mucopolysaccharidosis; the nerve metabolism disease; Niemann-Pick disease (niemann-pick); organic acidemia; purine; phenylketonuria (PKU); Pang Pu disease (pompe); false hurler syndrome (pseudo-hurler); pyruvate dehydrogenase deficiency; Sang Huofu disease (sandhoff); Sang Feilibo syndrome (sanfilippo); husky her syndrome (scheie); this bad syndrome (sly); Tai-Sa Ke disease (tay-sachs); front three aminuria disease (fish stench syndrome); the urea cycle disease; the vitamin D deficiency rickets; the muscle metabolism disease; the heredity dysbolism; acid-base imbalance; acidosis; alkalosis; alkaptonuria; α-mannosidosis; amyloidosis; anaemia; asiderosis; ascorbic acid deficiency; vitamin-deficiency; athlete's foot; biotinidase deficiency; lack glycoprotein syndrome; the carnitine obstacle; cystinosis; cystinuria; Fabry disease; the fatty acid oxidation obstacle; fucosidosis; galactosemia; Gaucher disease; gill uncle sick (gilbert disease); glucosephosphate dehydrogenase deficiency; glutaric acidemia (glutaric academia); glycogen storage disease; Hartnup disease (hartnupdisease); hemochromatosis; hemosiderosis; hepatolenticular degeneration; histidinemia; homocystinuria; hyperbilirubinemia; hypercalcinemia; hyperinsulinemia; potassemia; hyperlipidemia; hyperoxaluria; hypervitaminosis A; hypocalcemia; hypoglycemia; hypopotassaemia; hyponatremia; hypophosphatasia (hypophosphotasia); insulin resistance; iodine deficiency; the iron overload; chronic idiopathic jaundice; Leigh disease; Lai-Ni syndrome (Lesch-Nyhan); the leucine dysbolism; lysosomal storage disease; magnesium lacks; maple syrup urine disease; MELAS syndrome; Men Keshi Menkes (menkes kinky hair syndrome); metabolic syndrome X; mucolipidosis; mucopolysaccharidosis (Mucopolysacchabridosis); Niemann-Pick disease; fat; Ornithine carbamoyltransferase deficiency disease; malacosteon; pellagra; the peroxidase obstacle; porpharia; erythropoietic porphyria; senium praecox; false Gaucher disease; refsum's disease (refsumdisease); thunder last of the twelve Earthly Branches syndrome (reye syndrome); rickets; the Sang Huofu disease; Tangier disease (tangier disease); Tai-Sa Ke disease; the tetrahydrobiopterin defective; front three aminuria disease (fish stench syndrome); tyrosinemia; urea cycle disorder; water-EI; Wernike's encephalopathy (wernicke encephalopathy); VAD; vitamin B12 deficiency; Vitamin B deficiency; Wallman disease (wolman disease) and cerebro-hepato-renal syndrome (zellwegersyndrome).

Some preferred embodiment in, metabolic disease comprises diabetes and obesity.

The example of blood-lymphatic system

Blood-lymphatic system comprises blood and lymphopathy." hematologic effects " comprises disease, obstacle or the situation that influences hematopoietic cell or tissue.Hematologic effects comprises and unusual blood content or function diseases associated, obstacle or situation.The example of hematologic effects comprises: by the obstacle that marrow radiates or chemotherapy causes of cancer, following obstacle: as pernicious anaemia, hemorrhagic anemia, hemolytic anemia, alpastic anemia, sickle cell anemia, sideroblastic anemia, with such as malaria, trypanosomiasis, HIV, the anaemia that hepatitis virus or other viral chronic infection are relevant, the myelophthisic anemia that causes by the marrow defective, the kidney failure that causes by anaemia, anaemia, polycythemia (polycethemia), infectious mononucleosis (IM), acute nonlymphocytic leukemia (ANLL), acute myelogenous leukemia (AML), acute promyelocytic leukemia (APL), acute myelomonocytic leukemia (AMMoL), polycythemia vera (polycethemia vera), lymthoma, acute lymphatic leukemia (ALL), chronic lymphocytic leukemia, wilms' tumor, Ewing's sarcoma, retinoblastoma, hemophilia, increase relevant obstacle with the thrombosis risk, bleb, thalassemia (thalessemia), antibody-mediated obstacle such as transfusion reaction and erythroblastosis, such as microangiopathic hemolytic anemia, the red blood cell mechanical trauma of thrombotic thrombocytopenic purpura and disseminated intravascular coagulation, such as plasmodial parasitic infection, chemical damage and the hypersplenism that causes by saturnism for example.

Lymphatic disease includes, but are not limited to: lymphnoditis, lymphangiectasis, angioleucitis, lymphedema, lymphocyst, lymphoproliferative disorder, mucocutaneous lymph node syndrome, reticuloendotheliosis, splenius disease, thymic hyperplasia, thymic tumor, tuberculosis, lymphadenopathy, pseudolymphoma and lymph are unusual.

Some preferred embodiment in, the blood-lymphatic system obstacle comprises: non_hodgkin lymphoma, chronic lymphocytic leukemia and reactive lymphoid hyperplasia.

The example of CNS disease

The example of CNS disease includes, but are not limited to: the demyelinate of neurodegenerative disease, the drug abuse such as cocaine abuse, multiple sclerosis, schizophrenia, acute diseminated encephalomyelitis, transverse myelitis, demyelinate genetic disease, spinal cord injury, virus induction, carrying out property many focuses leukoencephalopathy (progressive multifocal leucoencephalopathy), myelopathy and the Nutrition and Metabolism obstacle relevant with human close lymph T cell virus I (HTLVI).

Some preferred embodiment in, the CNS disease comprises Parkinson's, Alzheimer disease, cocaine abuse and schizophrenia.

The example of neurodegenerative disease

The example of the neurodegenerative disease in the method for the present invention includes, but are not limited to: Alzheimer disease; Pick disease (Pick ' s disease); diffusivity thunder dimension body disease (diffuse lewy bodydisease); paralysis (Si-Li syndrome (Steel-Richardson syndrome)) on the carrying out property nuclear; multisystem degeneration (Xia-De Lei syndrome (Shy-Drager syndrome)); comprise amyotrophic lateral sclerosis; the degeneration incoordination; corticobasal degeneration; Guam ALS-Parkinson's-dull-witted compound disease (ALS-Parkinson ' s-dementia complex of guam); subacute sclerosing panencephalitis; Huntington (Huntington ' s disease); Parkinson's; touch nucleoprotein disease (synucleinopathies); carrying out property of primary aphasia; SND; Ma-Yue disease (Machado-Joseph disease)/3 type spinocebellar ataxia and olivopontocerebellar degeneration's motor neuron disease; (Gilles De La Tourette ' sdisease) for day Lars moral La Tulatai disease; bulbar paralysis and pseudobulbar paralysis; spinal cord and spinal cord oblongata amyotrophia (Kennedy's disease (Kennedy ' s disease)); primary lateral sclerosis; familial spastic; Wei-Huo disease (Werdnig-Hoffmann disease); Ku-Wei disease (Kugelberg-Welanderdisease); Tai-Sa Ke disease (Tay-Sach ' s disease); the Sang Huofu disease; the familial spastic disease; Wohlfart-Kugelberg-Welander disease (Wohlfart-Kugelberg-Welander disease); spastic paraparesis; carrying out property many focuses property leukoencephalopathy and prion disease (comprise Creutzfeldt-Jakob disease (Creutzfeldt-Jakob); Ge-Shi-Sha disease (Gerstmann-Straussler-Scheinker); kuru and the insomnia of mortality familial); Alexander disease (Alexander disease); A Erpo disease (alper ' s disease); amyotrophic lateral sclerosis; ataxia-telangiectasia; the Batten disease; Ka Nafan disease (canavan disease); neil-ingwall syndrome (cockayne syndrome); corticobasal degeneration; Creutzfeldt-Jakob disease; Huntington; Kennedy's disease; krabbe's disease; thunder dimension body dementia (lewy body dementia); Ma-Yue disease; 3 type spinocebellar ataxias; multiple sclerosis; multiple system atrophy; Parkinson's; Pelizaeus (Pelizaeus-Merzbacher Disease); refsum's disease; adrenoleukodystrophy (Schilder ' sdisease); the Spielmeyer-Vogt-Sjogren-Batten disease; Si-Li-Ao disease (Steele-Richardson-Olszewski disease) and tabetic crisis.

The example of urethra obstacle

The example of the urethra obstacle in the method for the present invention includes, but are not limited to: the obstacle of kidney, ureter, bladder and urethra.For example, urethritis, cystitis, pyelonephritis, renal aplasia, uronephrosis, multicystic kidney disease, polycystic kindey, down in urethral obstruction, ectopia vesicae and anaspadias, hypospadia, bacteruria disease, prostatitis, the kidney and peripheral abscess, benign prostatauxe, clear-cell carcinoma, transitional cell carcinoma, wilms' tumor, uremia and glomerulonephritis (glomerolonephritis).

The example of respiratory disease

Respiratory disease and situation comprise, but be not limited to: asthma, chronic obstructive pulmonary disease (COPD), gland cancer, the gland carcinoma squamosum, squamous cell carcinoma, large cell carcinoma, cystic fibrosis (CF), expiratory dyspnea (Dispnea), pulmonary emphysema, stridulate, pulmonary hypertension, pulmonary fibrosis, air flue excessively responds, adenosine or adenosine receptor level raise, the pulmonary branches tracheae shrinks, lung inflammation and allergic reaction, exhaust with surface reactive material, chronic bronchitis, bronchoconstriction, expiratory dyspnea, the lung airway that hinders and block, adenosine test for cardiac function, the lung vessel retraction, breathing is obstructed, acute respiratory distress syndrome (ARDS), use such as the some drugs of the medicine that improves adenosine and adenosine level and for example treat the other medicines of supraventricular tachycardia (SVT) and carry out adenosine and stress test, infant respiratory distress syndrome (baby RDS), pain, allergic rhinitis, pulmonary surfactant reduces, reduction of ubiquinone level or chronic bronchitis etc.

The example of female reproductive system obstacle

The female reproductive system obstacle comprises the disease of vulva, vagina, cervix, corpus uteri, fallopian tubal and ovary.Some example comprises, such as the oviduct disease, disease of ovary, liomyoma, MCAC, serous cystadenocarcinoma, the adnexal disease of parovarian cyst and pelvic inflammatory disease, mullerianosis, such as the fallopian tubal tumour, cervix tumor, vaginal tumor, the reproductive system tumour of tumor of vulva and ovarian neoplasm, ankylocolpos, the reproduction bleb, infertile, sex dysfunction such as dyspareunia and impotence, tuberculosis, such as cervix diseases, endometrial hyperplasia, endometritis, hemometra, uterine hemorrhage, cervix tumor, the prolapse of uterus, the uterine disease of hysterorrhexis and inversion of uterus, such as dyspareunia, hematocolpos, vaginal fistula, vaginal tumor, vaginitis, the vaginal disease of vaginal fluid and candidiasis or vulvovaginal disease, such as kraurosis vulvae, itch, tumor of vulva, vulvitis and oidiomycotic vulva disease, urogenital disease with and urogenital tumour unusual such as urogenital.

The example of male reproductive system obstacle

The male reproductive system obstacle includes, but are not limited to: epididymitis; such as penile tumor; the reproductive system tumour of tumor of prostate and orchioncus; vaginal hematocele; the reproduction bleb; hydrocele; sterile; such as balanitis; hypospadia; fibrous cavernositis of penis (peyronie disease); penile tumor; the penile disease of phimosis and priapism; such as hypertrophy of the prostate; tumor of prostate and prostatitic prostatic disorders; organic sex dysfunction such as dyspareunia and impotence; torsion of spermatic cord; seminal cyst; such as cryptorchidism; the disease of testis of orchitis and orchioncus; tuberculosis; varicocele; the urogenital disease and the Fournier gangrene of and urogenital tumour unusual such as urogenital.

The example of cardiovascular disorder (CVS)

Cardiovascular disorder comprises that those can cause ischaemic or be poured into the obstacle that causes by heart again.Example comprises, but be not limited to: the relevant situation of atherosclerotic, coronary artery disease, granulomatous myocarditis, chronic myocarditis (non-granulomatous), primary hypertrophic cardiomyopathy, peripheral arterial disease (PAD), apoplexy, angina pectoris, miocardial infarction, the cardiovascular tissue damage that causes by asystole, cardiovascular tissue damage, cardiogenic shock and those of ordinary skills dysfunction known or that relate to heart or vascular system that causes by heart bypass or tissue damage (especially, but be not limited to and PARP activates relevant tissue damage).

Of the present invention some preferred embodiment in, the CVS disease comprises: atherosclerotic, granulomatous myocarditis, miocardial infarction, be secondary to the myocardial fibrosis of valvular heart disease, myocardial fibrosis, primary hypertrophic cardiomyopathy and the chronic myocarditis (non-granulomatous) that form of infraction.

Method with the PARP inhibitor for treating

When the PARP inhibitor is used for treatment such as the various diseases of myocardial ischemia, apoplexy, head injury and neurodegenerative disease independently, with as the supplemental treatment of other medicament that comprises chemotherapeutics, radiation, oligonucleotides or antibody in the treatment of cancer time, has potential treatment benefit.Scope of the present invention without limits should be appreciated that various PARP inhibitor are known in the art, and within the scope of the present invention.Herein disclosed is some examples of PARP inhibitor, but they limit the scope of the invention absolutely not.

Most of PARP inhibitor has been designed to the analog of benzamide, its in the catalytic site of PARP competitively in conjunction with natural substrate NAD.The PARP inhibitor comprises, but is not limited to: benzamide, quinolone and isoquinolone, benzopyrone, 3,5-two iodo-4-(4 '-the methoxyl phenoxy group) methyl benzoate and 3,5-two iodo-4-(4 '-methoxyl-3 ', 5 '-two iodo-phenoxy groups) methyl benzoate (US5,464,871, US5,670,518, US6,004,978, US6,169,104, US5,922,775, US6,017,958, US5,736,576 and US5,484,951, the complete introducing of this paper).The PARP inhibitor comprises the various cyclohexyl benzamide analogs (that is lactams) of the potent inhibitor that is the NAD site.Other PARP inhibitor comprises, but be not limited to: benzimidazole and indoles (EP841924, EP1127052, US 6,100,283, US6,310,082, US2002/156050, US2005/054631, WO05/012305, WO99/11628 and US2002/028815).Used many low-molecular-weight PARP inhibitor to illustrate the function of poly-ADP ribosylation in DNA repairs.In the cell of handling with alkylating agent, the inhibition of PARP causes DNA bound rupture and cell killing showed increased (people such as Durkacz, 1980, Nature 283:593-596; And Berger, N.A., 1985, Radiation Research, 101:4-14).Afterwards, shown that such inhibitor had improved effect (people such as Ben-Hur, 1984, British Journal of Cancer, 49 (Suppl.VI): the 34-42 of radioreaction by the reparation that suppresses PLD; With people such as Schlicker, 1999, Int.J.Radiat.Bioi., 75:91-100).Reported that the PARP inhibitor is effective (US5,032,617,5,215,738 and 5,041,653) for radiosensible anoxic tumour cell.In addition, (PARP-/-) animal response alkylating agent that PARP knocks out and γ-radiation show genomic instability (people such as Wang, 1995, Genes Dev., 9:509-520; With people such as Menissier de Murcia, 1997, Proc.Natl.Acad.Sci.USA, 94:7303-7307).

The PARP inhibitor studies show that the oxygen radical dna damage (it is discerned by PARP subsequently) that causes the DNA bound rupture is main contribution factor (people such as Cosi, 1994, J.Neurosci.Res., the 39:38-46 for such morbid state; With people such as Said, 1996, Proc.Natl.Acad.Sci.U.S.A., 93:4688-4692).The retroviral infection that has also proved effective mammalian cell is blocked by the inhibition of PARP activity.Prove that (people such as Gaken, 1996, J.Virology, 70 (6): 3992-4000) appear in being suppressed in the various cell type that such recombinant retroviral vector infects.Therefore, developed the PARP inhibitor (WO91/18591) that is used for antiviral therapy and treatment of cancer.And, infer that PARP suppresses to have postponed the human fibroblast and aging character (Rattan and Clark, 1994, Biochem.Biophys.Res.Comm., 201 (2): 665-672) occur.Effect relevant (d ' people such as Addadi Fagagna, 1999, Nature Gen., 23 (1): 76-80) that this may bring into play in control telomere function with PARP.

The PARP inhibitor has following architectural feature: 1) acid amides or lactams functional group; 2) the NH proton of this acid amides or lactams functional group keeps with effective bonding; 3) be connected on the aromatic rings amide group or with aromatic rings thick and the lactams group; 4) the best cis-configuration of the acid amides in the fragrant plane; With 5) single aryl carboxamides is limited in the assorted polycyclic lactams (people such as Costantino, 2001, J Med Chem., 44:3786-3794).People such as Virag, 2002, Pharmacol Rev., 54:375-429,2002 have summed up various PARP inhibitor.Some example of PARP inhibitor comprises, but be not limited to: isoquinolone and dihydro isoquinolone are (for example, US 6,664,269 and WO99/11624), niacinamide, 3-aminobenzamide, single aryl amide and bicyclic lactam, tricyclic lactam or tetracyclic lactam, phenanthridone (people such as Perkins, 2001, Cancer Res., 61:4175-4183), 3,4-dihydro-5-methyl-isoquinoline-1 (2H)-ketone and benzoxazole-4-formamide (people such as Griffin, 1995, Anticancer Drug Des, 10:507-514; People such as Griffin, 1998, J Med Chem, 41:5247-5256; With people such as Griffin, 1996, Pharm Sci, 2:43-48), dihydro-isoquinoline-1 (2H)-ketone, 1,6-benzodiazine-5 (6H)-ketone, quinazoline-4 (3H)-ketone, thieno [3,4-c] pyridine-4 (5H) ketone and thieno [3,4-d] pyrimidine-4 (3H) ketone, 1,5-dihydroxy isoquinoline and 2-methyl-quinazoline-4[3H]-ketone (people such as Yoshida, 1991, J Antibiot (Tokyo) 44:111-112; People such as Watson, 1998, Bioorg Med Chem., 6:721-734; With people such as White, 2000, J Med Chem., 43:4084-4097), 1,8-naphthalimide derivative and (5H) phenanthridines-6-ketone (people such as Banasik, 1992, J Biol Chem, 267:1569-1575; People such as Watson, 1998, Bioorg Med Chem., 6:721-734; People such as Soriano, 2001, Nat Med., 7:108-113; People such as Li, 2001, Bioorg Med Chem Lett., 11:1687-1690; With people such as Jagtap, 2002, Crit Care Med., 30:1071-1082), tetracyclic lactam, 1,11b-dihydro-[2H] benzopyranyl [4,3,2-de] isoquinoline-3-ketone, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (people such as Zhang, 2000, Biochem Biophys Res Commun., 278:590-598; With people such as Mazzon, 2001, Eur J Pharmacol, 415:85-94).Other example of PARP inhibitor includes, but not limited to describe in the following patent: US 5,719, and 151, US 5,756,510, US 6,015, and 827, US 6,100, and 283, US 6,156,739, US 6,310, and 082, US 6,316, and 455, US 6,121,278, US 6,201, and 020, US 6,235, and 748,6,306,889, US 6,346, and 536, US 6,380,193, US 6,387, and 902, US 6,395, and 749, US 6,426,415, US 6,514, and 983, US 6,723,733, US 6,448, and 271, US 6,495, and 541, US 6,548,494, US 6,500, and 823, US 6,664,269, US 6,677, and 333, US 6,903, and 098, US 6,924,284, US 6,989, and 388, US 6,277,990, US 6,476, and 048 and US 6,531,464.The other example of PARP inhibitor comprises, but be not limited to, describe in the open text of following patented claim: US 2004198693A1, US2004034078A1, US 2004248879A1, US 2004249841A1, US2006074073A1, US 2006100198A1, US 2004077667A1, US2005080096A1, US 2005171101A1, US 2005054631A1, WO 05054201A1, WO 05054209A1, WO 05054210A1, WO 05058843A1, WO 06003146A1, WO 06003147A1, WO 06003148A1, WO 06003150A1 and WO05097750A1.

In one embodiment of the invention, the PARP inhibitor is compound and pharmaceutically acceptable salt, solvate, isomers, dynamic isomer, metabolin, analog or the prodrug of formula (Ia):

Wherein, R ₁, R ₂, R ₃, R ₄And R ₅Be independently selected from hydrogen, hydroxyl, amino, nitro, iodine, (C ₁-C ₆) alkyl, (C ₁-C ₆) alkoxy, (C ₃-C ₇) naphthenic base and phenyl, wherein, 5 R ₁, R ₂, R ₃, R ₄And R ₅At least two in the substituting group are always hydrogen, and at least one in 5 substituting groups is always nitro, and substituting group of at least one contiguous nitro is always iodine.R ₁, R ₂, R ₃, R ₄And R ₅It also can be halogen such as chlorine, fluorine or bromine.United States Patent (USP) 5 464 871 provides the further details about the compound of formula Ia.

The compound of a kind of preferred formula Ia is according to the compound of formula Ia and pharmaceutically acceptable salt thereof:

Wherein, R ₂, R ₃, R ₄And R ₅Be independently selected from hydrogen, hydroxyl, amino, nitro, iodine, (C ₁-C ₆) alkyl, (C ₁-C ₆) alkoxy, (C ₃-C ₇) naphthenic base and phenyl, wherein, 5 R ₁, R ₂, R ₃, R ₄And R ₅At least two in the substituting group are always hydrogen, and in 5 substituting groups at least one is always nitro.

The compound of a kind of preferred formula Ia is:

4-iodo-3-nitrobenzamide

(BA)。

In some embodiments, the chromone compound of formula II is used for method of the present invention.The chromone compound of formula II is compound or its pharmaceutically acceptable salt, solvate, isomers, dynamic isomer, metabolin or the prodrug that is shown below:

Formula II

Wherein, R ₁, R ₂, R ₃And R ₄Be independently selected from H, halogen, the optional hydroxyl that replaces, the optional amine that replaces, the optional low alkyl group that replaces, the optional phenyl that replaces, the optional C that replaces ₄-C ₁₀Heteroaryl, the optional C that replaces ₃-C ₈Naphthenic base (No. 5484951, the complete introducing United States Patent (USP) of this paper).

Some embodiment adopts compound and the pharmaceutically acceptable salt thereof with following chemical formula:

Wherein, R ₁, R ₂, R ₃Or R ₄Be selected from hydrogen, hydroxyl, amino, (C independently of one another ₁-C ₆) alkyl, (C ₁-C ₆) alkoxy, (C ₃-C ₇) naphthenic base, halogen and phenyl, wherein, 4 R ₁, R ₂, R ₃Or R ₄At least three in the substituting group are always hydrogen.

Some embodiment adopts compound and the pharmaceutically acceptable salt thereof with following chemical formula:

Wherein, R ₁, R ₂, R ₃Or R ₄Be selected from hydrogen, hydroxyl, amino, (C independently of one another ₁-C ₆) alkyl, (C ₁-C ₆) alkoxy, (C ₃-C ₇) naphthenic base, halogen and phenyl, wherein, 4 R ₁, R ₂, R ₃Or R ₄At least three in the substituting group are always hydrogen.

Some embodiment adopts the compound with following chemical formula:

Wherein, R ₁, R ₂, R ₃Or R ₄Be selected from hydrogen, hydroxyl, amino, (C independently of one another ₁-C ₆) alkyl, (C ₁-C ₆) alkoxy, (C ₃-C ₇) naphthenic base, halogen and phenyl, wherein, 4 R ₁, R ₂, R ₃Or R ₄At least three in the substituting group are always hydrogen.

A kind of preferred embodiment in, the present invention relates to the chromone compound of following formula II:

6-amino-5-iodo-benzopyrone

(BP)

In other more a kind of embodiment, the compound that is used for method as herein described is:

United States Patent (USP) 5 484 951 provides the further details about chromone compound, and this paper is complete to be incorporated herein by reference.

Below be possible: the most powerful and potent PARP inhibitors (promptly, the material standed for that drug development is possible) can't from scientific literature, obtain, but accepting clinical testing or may finally come across disclosed patent and the various databases of unsettled patented claim in.All such PARP inhibitor within the scope of the present invention.Except to PARP optionally, powerful enzyme suppresses, and can also adopt some other methods to suppress the cytoactive of PARP in cell or in animal used as test.The inhibition that intracellular Ca2+ is mobilized prevents that the PARP that oxygenant is induced from activating, NAD+ exhausts and meronecrosis, this is thymocyte (people such as Virag, 1999, MolPharmacol., 56:824-833) and enterocyte (people such as Karczewski, 1999, BiochemPharmacol. obtains proof in 57:19-26).Shown to be similar to calcium chelating agent, intracellular zinc sequestrant prevent the PARP activation of oxygenant mediation and meronecrosis (people such as Virag, 1999, BrJ Pharmacol., 126:769-777).Intracellular purine (inosine, hypoxanthine), except bringing into play various effects, also can be used as PARP inhibitor performance biological action (people such as Virag, 2001, FASEB J., 15:99-107).

Method provided by the invention can comprise to be used PARP inhibitor itself or unites other treatment.Situation to be treated is partly depended in the selection of the treatment that can use altogether with composition of the present invention.For example, in order to treat acute myelogenous leukemia, the compound of some embodiment of the present invention can use with radiotherapy, monoclonal antibody therapy, chemotherapy, transplantation of bone marrow or its combinatorial association.

To patient, preferred mammal, the more preferably PARP inhibitor of human administration effective therapeutic dose disclosed herein, comprise that with influence the PARP enzyme suppresses or the active pharmacologically active that suppresses of PARP.Like this, PARP inhibitor of the present invention can be used for treating or preventing the multiple disease of animal, comprises because cellular damage that necrosis or apoptosis cause or dead neural tissue injury, cerebral ischemia and reperfusion injury or the neurodegenerative disease that causes.In addition, compound of the present invention also can be used for the cardiovascular disorder for the treatment of animal by PARP inhibitor from effective dose to animal that use.In addition, compound of the present invention can be used for the treatment of cancer, and makes tumour cell for radiation-sensitive or chemical-sensitive.

In some embodiments of the present invention, the PARP inhibitor can be used for regulating the neuron of damage, promotes neuron regeneration, prevention nerve degeneration and/or treatment neurological disorder.The PARP inhibitor suppresses the PARP activity, thereby, the damage that the neural tissue injury that can be used for treating animal, especially cancer, angiocardiopathy, cerebral ischemia and reperfusion injury or neurodegenerative disease cause.PARP inhibitor of the present invention can be used for treating patient's damage to cardiac tissue, the especially damage that causes of heart ischemia or reperfusion injury.Compound of the present invention especially can be used for treating the cardiovascular disorder that is selected from coronary artery disease such as atherosclerotic, angina pectoris, miocardial infarction, myocardial ischemia and asystole, heart bypass and cardiogenic shock.

On the other hand, PARP inhibitor of the present invention can be used for treating cancer, or combination chemotherapy, radiotherapy or radiation.PARP inhibitor of the present invention is " anticancer ", and this term also comprises " antitumor cell growth stimulator " and " antitumor agent ".For example, PARP inhibitor of the present invention can be used for the treatment of cancer, and makes the tumour cell of cancer for radiation-sensitive and/or chemical-sensitive.

Known radiosensitizer can increase the susceptibility of cancer cell for the poisonous effect of electromagnetic radiation.Many modality of cancer treatment adopt the radiosensitizer by the electromagnetic radiation activation of X-ray at present.The example of the radiosensitizer of X-ray activation includes, but are not limited to following: metronidazole, Misonidazole, desmethylmisonidazole (desmethylmisonidazole), Pimonidazole, etanidazole, Nimorazole, mitomycin C, RSU 1069, SR 4233, EO9, RB 6145, niacinamide, 5-bromouracil deoxyribose (BUdR), 5-iododeoxyuridine (IUdR), bromine deoxycytidine (Bromodeoxycytidine), fluoro BrdU (fluorodeoxyuridine) (FudR), hydroxycarbamide, the analog and the derivant of the same compound of cis-platinum and treatment effective dose.

The photodynamic therapy of cancer (PDT) adopts the radioactivation agent of visible light as sensitizing agent.The example of photodynamics radiosensitizer includes, but are not limited to: the analog and the derivant of the same compound of hematoporphyrin derivative, phytochrome (photofrin), benzoporphyrin derivative, NPe6, etioporphyrin SnET2, pheoborbide-α, bacteriochlorophyll-α, naphthalene phthalocyanine, phthalocyanine, zinc phthalocyanine and treatment effective dose.

Radiosensitizer can be used with one or more other PARP inhibitor of treatment effective dose, these PARP inhibitor comprise, but are not limited to: promote radiosensitizer to be attached to the PARP inhibitor that PARP inhibitor, control therapeutic agent, nutrients and/or oxygen in the target cell flow to target cell.Similarly, also the known chemical sensitizing agent increases the susceptibility of cancer cell for the poisonous effect of chemotherapy compound.Can include, but are not limited to the exemplary chemotherapeutant of PARP inhibitor use in conjunction: adriamycin (adriamycin), camptothecine, Dacarbazine, carboplatin, cis-platinum, daunorubicin, docetaxel (docetaxel), Doxorubicin (doxorubicin), interferon (α, β, γ), interleukin-22, Erie replace the analog and the derivant of the same compound of health (innotecan), taxol (paclitaxel), streptozotocin, Temozolomide (temozolomide), Hycamtin (topotecan) and treatment effective dose.In addition, can comprising of other with the therapeutic agent that the PARP inhibitor is united use, but be not limited to: 5 FU 5 fluorouracil, formyl tetrahydrofolic acid, 5 '-amino-5 '-deoxyribosylthymine, oxygen, carbogenes (carbogen), red blood cell transfusion, perfluocarbon are (for example, Fluosol-DA), 2,3-DPG, BW12C, calcium channel blocker, oxpentifylline (pentoxyfylline), anti-angiogenic compounds, hydrolazine and L-BSO.

In some embodiments, the therapeutic agent that is used for the treatment of comprises in conjunction with the antibody of PARP or reagent, thereby reduces experimenter's PARP level.In other embodiments, for PARP level and/or the PARP activity that influences the experimenter, can regulate the expression of cell.Can use transgenosis and gene therapy technology to come polynucleotide molecule delivery of therapeutic and/or preventative.Thereby thereby other reagent comprise of the present invention interact to influence the micromolecule of its function and to interact in conjunction with PARP or with PARP in conjunction with the nucleotide sequence of coding PARP or with the nucleotide sequence of coding PARP influence the micromolecule of PARP level of the present invention.In order to treat disease, these reagent can be used separately or with known and be that the treatment of available other type is co-administered for those skilled in the art.In some embodiments, the PARP inhibitor that is used for the treatment of can be curative or preventative, or the both is.The PARP inhibitor can directly act on PARP, or regulates other the cell component influence the PARP level subsequently.Some preferred embodiment in, the PARP inhibitor suppresses the activity of PARP.

Methods of treatment disclosed herein can by in oral administration, mucosal, oral administration, intranasal administration, suction, parenteral, intravenous, subcutaneous, the muscle, hypogloeeis, percutaneous dosing, carry out through ophthalmic administration and rectally.

Be adapted at determining that experimenter's disease can comprise with the pharmaceutical composition of the PARP inhibitor that is used for the treatment of after the PARP inhibitor for treating, the pharmaceutical composition that wherein comprises the active component of treatment or prevention effective dose (that is, effectively obtaining the content of treatment or prevention benefit).The actual effective dose that is used for application-specific especially depends on state to be treated and route of administration.Within definite complete those skilled in the art's of effective dose the ability.Pharmaceutical composition comprises PARP inhibitor, one or more pharmaceutically acceptable carriers, thinning agent or excipient and optional other therapeutic agent.Composition can be made and continue to discharge or delayed release preparation.

Can use composition by injection, local, oral, transdermal, rectum or through suction.The oral form of administering therapeutic agent comprises powder, tablet, capsule, solution or emulsion.Can use effective dose with single dose or by the serial dosage of separating at interval such as the appropriate time of a few hours.The compounding pharmaceutical composition uses the acceptable carrier that comprises excipient and assistant agent on one or more physiology in a usual manner, and it helps reactive compound is processed as can be at the preparation that pharmaceutically uses.Appropriate formulation depends on the method for application of selection.The suitable technique for preparing the pharmaceutical composition of therapeutic agent of the present invention is to know in this area.

A kind of preferred dosage of 4-iodo-3-nitrobenzamide is since first day, biweekly, is surpassing in one hour time intravenous injection 4mg/kg (dosage of 4-iodo-3-nitrobenzamide preferably at least at interval two days).The treatment of 4-iodo-3-nitrobenzamide preferably biweekly, continuous three all venoclysises in each cycle of 28 days.Other preferred dosage comprises 0.5,1.0,1.4,2.8 and 4mg/kg as single therapy or therapeutic alliance.

Should be appreciated that reactive compound and the proper dosage that comprises the composition of reactive compound can change with the patient.Determine that optimal dose generally comprises treatment benefit level and any risk or harmful spinoff of balance treatment of the present invention.The dosage level of selecting depends on multiple factor, include but not limited to: the drainage rate of the activity of specific PARP inhibitor, method of administration, administration time, compound, the duration of treatment, the other medicines of uniting use, compound and/or material and patient's age, sex, body weight, situation, general health situation and former medical history.Although usually dosage can reach the local concentration of realizing required effect and not causing harmful in fact spinoff at action site, the amount of compound and method of administration finally depend on doctor's judgement.

Can in whole therapeutic process, carry out vivo medicine-feeding with a kind of dosage by continuously or intermittently (for example, the dosage of separating with proper spacing).The method of determining effective and efficient manner and dosage is that those skilled in the art is known, and can change along with the formulation that is used for the treatment of, therapeutic purposes, the target cell of being treated and the experimenter who is treated.Can carry out the single or multiple administration according to dosage level and the pattern selected by the treatment doctor.

The treatment standard of cancer location

In another aspect of this invention, the PARP inhibitor uses with the base therapy standard association of the cancer that is used for being treated.This paper has described the treatment for cancer standard to some type.In some embodiments, PARP inhibitor and treatment standard association described herein are used.

Endometrium

The base therapy standard that four kinds of treatment carcinomas of endometrium are arranged comprises operation (total hysterectomy inside fascia removes art, bilateral S﹠O and radical hysterectomy), radiotherapy, chemotherapy and hormone therapy.In some cases, use the supplemental treatment relevant with described treatment.

Mammary gland

The treatment of breast cancer comprise at present use or do not use Tamoxifen the reservation operation on breast with radiation therapy, use or do not use the total mastectomy of Tamoxifen, do not carry out radiocurable reservation operation on breast, do not carry out the nodi lymphatici axillares excision the preventative total mastectomy of bilateral, send Tamoxifen and reduce the incidence of breast cancer subsequently and the supplemental treatment relevant with described treatment.

Ovary

If tumour is good or the appropriateness differentiation, removes art and be enough for the patient who is in early stage disease with the bilateral S﹠O of omentectomy through the belly total hysterectomy inside fascia.Be diagnosed as the patient who is in III phase and IV phase with operation and chemotherapy treatment.

Cervix

The method of treatment Exocervix damage comprises loop electrosurgical resection art (LEEP), laser therapy, conization and cold therapy.For the tumour of I phase and II phase, the treatment option comprises: total hysterectomy inside fascia removes art, conization, radical hysterectomy and independent intracavitary therapy, bilateral pelvic lymphadenectomy, the full pelvis radiation therapy in operation back and adds that chemotherapy and radiotherapy add the chemotherapy of using cis-platinum or cis-platinum/5-FU.For the tumour of III phase and IV phase, the treatment standard of cervix cancer is that radiotherapy and/or use comprise that cis-platinum, ifosfamide, ifosfamide-cis-platinum, taxol, Erie replace the chemotherapy of the medicine of health, taxol/cis-platinum and cis-platinum/gemcitabine.

Testis

Seminomatous treatment standard be to use or do not use single dose carboplatin supplemental treatment radical-ability groin orchiectomy, by radical-ability groin resection remove testis succeeded by radiation therapy and by radical-ability groin orchiectomy succeeded by chemotherapy or radiocurable combination for belly and pelvic lymph node.For nonseminomatous patient, treatment comprises that removing testis by the groin art removes succeeded by retroperitoneal lymph node, and chemotherapeutical radical-ability groin orchiectomy is removed, carried out or do not carry out to the retroperitoneal lymph node that carries out or do not carry out the retroperitoneal lymph node removal, carries out or do not keep fecundity.

Lung

In non-small cell lung cancer (NSCLC), except the cancer that localizes most, the result of standard care is relatively poor.The NSCLC patient of all new diagnosis is potential candidates of the research of the new form of therapy of assessment.Operation is to select for the treatment that the most probable of this disease is cured; Radiation therapy can produce to cure and provide in Most patients in a small amount of patient and alleviate.Auxiliary chemotherapy can provide extra benefit for the patient who excises NSCLC.In the disease, use chemotherapy late.

Skin

The classic method of basal-cell carcinoma treatment comprises uses freezing art, radiation therapy, electrodesiccation and curettage and single excision.The localization squamous cell carcinoma of skin is highly recoverable disease.Traditional methods of treatment comprises uses freezing art, radiation therapy, ED﹠C and single excision.

Liver

Hepatocellular carcinoma may be cured by excision, but operation just suffers from the patient's of local disease treatment selection for fraction.Other treatment still rests on the clinical research stage, comprises whole body or input chemotherapy, HAL or embolism, through skin ethanol injection, RF ablation, cold therapy and radiolabeled antibody, often combined surgery excision and/or radiation therapy.

Thyroid gland

The standard care option of thyroid cancer comprises total thyroidectomy, lobectomy and described operation and I ¹³¹Melt, the external beam radiation-therapy, suppress thyroid-stimulating hormone (TSH) and chemotherapeutic combination with thyroxine.

Esophagus

The base therapy method comprises independent operation or chemotherapy associating radiation therapy.In independent case, use the various combinations of the endoscope therapy of operation, chemotherapy, radiation therapy, support, photodynamic therapy and use Nd:YAG laser, can obtain effective alleviation.

Kidney

Excision is the main treatment means of this disease.Even in the patient who suffers from the dispersivity tumour, the treatment of regional area form plays an important role in the symptom of alleviating the generation of primary tumo(u)r or ectopic hormone.Whole body therapeutic shows to have only limited effect.

In one embodiment, the PARP inhibitor is united other chemotherapeutant,, improves respectively for the many treatment for cancer such as colorectal cancer and cancer of the stomach, melanoma and glioma for health, Hycamtin, cis-platinum or Temozolomide as Erie.In another embodiment, the colorectal cancer in late period is treated for health by PARP inhibitor associating Erie, or the associating Temozolomide is treated chromoma.

In the cancer patient, in one embodiment, the PARP inhibitor is used for improving radiotherapy and chemotherapeutic treatment benefit.In another embodiment, target PARP is used for preventing tumour DNA plerosis itself and develops drug resistance that this can be so that they be more responsive for treatment of cancer.In more other a kind of embodiment, the PARP inhibitor (for example is used for improving various chemotherapeutants, methylating reagent, DNA topoisomerase enzyme inhibitor, cis-platinum etc.) and radiation for the effect of the tumour (for example, glioma, melanoma, lymthoma, colorectal cancer, neck tumour) of wide spectrum.

Kit

In other again one side, the invention provides the kit that the disease that is used for definite experimenter can be used the PARP modulators for treatment, wherein, this kit can be used to detect the PARP level from the sample that the experimenter obtains.For example, kit can be used to PARP level and/or the PARP activity in definite normal structure as herein described and the pathological tissues, and wherein, the PARP level is different in sick patient and normal subjects's sample.In one embodiment, kit comprises substrate, contain adsorbent on the substrate, wherein, this adsorbent is suitable in conjunction with PARP and/or RNA, and comprise instructions, illustrate by adsorbent being contacted sample and detecting the PARP that adsorbent keeps and determine PARP and/or PARP level and/or PAR (the sugared and multinuclear sugar of monokaryon).In another embodiment, kit comprises: (a) specificity in conjunction with PARP or with the interactional reagent of PARP; (b) detectable.In some embodiments, kit may further include the instructions of the appropriate operational parameters of label or independent card format.Whether randomly, kit may further include standard or contrast information, makes specimen to compare with the contrast information standard, be the diagnosis amount with the detection limit of determining detected PARP in sample.

In some embodiments, the independent composition that also can be used as in the independent container within the kit that is used for the treatment of of therapeutic agent provides.Suitable packing and other use article (for example, be used for the liquid preparation measuring cup, be used to make air to contact minimized Foilpac etc.) be known in the art, and can be included in the kit.

Embodiment 1

The GeneChip array has been widely used for monitoring mRNA and has expressed in many fields of biomedical research.Because gene is differently expressed in tissue and cell, highdensity oligonucleotide arrays technology allows the researchist to monitor ten hundreds of genes in a hybrid experiment.The express spectra of the mRNA molecule by the combined strength information acquisition gene that obtains with the probe in the probe groups, probe groups to forming, is inquired after the different piece of gene order by the probe of 11-20 the length oligonucleotides that is 25bp.

Use Affymetrix human genome genetic chip (covering 45000 genetic transcription things of 28473 UniGene bunch) assessment expression of gene.Use the total RNA of the about 5 μ g in the every duplicate samples of transcript labelling kit mark of high yield, and (Santa Clara is CA) with RNA hybridization, washing and the scanning of mark for Affymetrix, Inc. according to the producer's instructions.Affymetrix Microarray Suite 5.0 softwares (MAS5) are used for the transcript signal level of assessment from scan image (Affymetrix).It is 500 that signal standards on each array is turned to truncation mean value, get rid of signal minimum 2% and the highest by 2%.Hereinafter for convenience, the Affymetrix probe groups of the Genbank sequence that representative is unique is called probe or gene.In order to check any mistake in the expression that causes by image deflects, measure the related coefficient of each array for ideal distribution, wherein, desirable distribution is the mean value of all arrays.Use is filtered gene by the detection P value of MAS5 report from the residue array.Leave out in 95% array and have P 0.065 gene, and carry out the statistical of classification with all other signals.

Embodiment 2

The expression of PARP1 mRNA in normal breast of the mankind and infitrating ductal carcinoma

Research and design

Identify normal breast and infitrating ductal carcinoma sample in the system, this system be for

The member of the sample sets of system's regulation.Also assess the number percent tumour of each tumor sample and explain (percent tumor annotation), it is by by the ratio of checking the pernicious and non-pernicious karyocyte that virologist's quantitative measurement exists from the section of handling the collection of sample adjacent on microscope slide.

Assessed altogether 237 independently samples in this research, sample number is with respect to each the IDC hypotype that occurs in the Table A.Table A has also shown in the sample number as each IDC hypotype of the number percent of the observed sample of tumor tissues.

Table A: the sample size of histological type and number percent tumour

Table A shows〉90% IDC sample comprises 50% or more tumor tissues, and about 2/3 in all IDC samples comprise 75% or more tumor tissues, shows that it is the good representative of being rich in tumor sample.

Should be noted that any IDC sample can occur in more than one hypotype group.Show an example at table among the B, shown the IDC sample of 7 selections and they appearance in a plurality of, single or 0 IDC hypotype.For example, sample GID 7273 is not classified as any single hypotype, therefore only as general IDC sample evaluating.7287 of sample GID are classified as a hypotype, thereby for the result of its II phase classification and general IDC classification contribution are arranged.Sample GID 7387 is classified as two hypotypes, thereby for the result of these two hypotypes and general IDC classification contribution is arranged.

Table B: for the example of the hypotype classification of the IDC sample of selecting

On the HG-U133A array, be that the single probe sets of " W208644_at " is represented the PARP1 gene by identifier.All results in this report are based on for the MASS expression signal intensity of this probe sets and produce, and are called as ＂ PARP1 ＂.

All sample sets statistical study

Normal and IDC summarizes statistics

Normal and general IDC sample classification is pressed mean value, standard deviation, standard error and several confidence upper limit that distributes based on t and is summarized.Confidence upper limit (UCL) is similar to the standard deviation statistics, because they determine the specific region for the probability of observing a value.For example, 95% confidence upper limit is similar to the value predicted of having an opportunity in 5% sample.

For the normal data of breast, sample number (n=68) is enough big, makes t distribute and extremely be similar to the result who obtains when only using standard deviation to set limit.For example, the mean value+2SD of normal breast expression intensity is 365.06, and it is very similar to 95% confidence limit 365.92.The organ that the normal specimens number is lower is not like this.

Table C shows the summary statistics for each normal breast and general IDC sample sets.

Table C: the summary statistics of normal and general IDC breast sample sets

Group	N	Mean value	Standard deviation	Standard error		90％UCL	95％UCL	99％UCL	99.9％UCL
										Infitrating ductal carcinoma	169	328.487	135.695	10.4381	553.586	597.166	683.073	784.324
Normal structure	68	201.780	81.636	9.8998	338.939	365.919	419.800	484.808

IDC mean value/normal mean value=1.63

T-check for (IDC mean value=normal mean value) produces p=6*10 ^-16

Therefore, when IDC was moderate with respect to the change multiple of normal specimens, change had very high conspicuousness.

The assessment of single sample

Then, with respect to normal breast sample distribution, single test is from the single sample of general IDC breast sample sets He all IDC hypotypes.90%, 95%, 99% and 99.9% the confidence upper limit that each is defined as surpassing.Neither one IDC sample is lower than 90% confidence lower limit 64.6, so do not propose the LCL boundary.

Fig. 4 a shows the result's of each breast sample classification visual summary.Each cruciform represents to meet the hypotype of x-axle demonstration and a sample of the expression intensity that the y-axle shows.In addition, each point comes painted by tumour number percent intrinsic in the sample.Fig. 4 b is identical with Fig. 4 a, except the highest sample within the IDC grouping has been removed to allow better demarcation.

Result based on Fig. 4 a is:

The expression of the height of PARP1 is tangible with respect to normal breast sample in the IDC breast sample.

PARP1 in the IDC breast sample expresses and shows the variation (that is, bigger diffusion) of expressing much higher degree than the PARP1 in the normal breast sample.

As if two normal breast samples have higher PARP1 expression intensity than other 66 samples, and be not the part of identical distribution.

As if an IDC breast sample has very high expression intensity, and be not the part of identical distribution.

At least as if from range estimation, tumour number percent can the expression intensity of high level ground influence in IDC breast sample.

Table D has summarized number percent and the quantity that surpasses for the sample of IDC classification and the predefined confidence upper limit of hypotype thereof.

The number percent and the quantity of the sample that surpasses UCL of table D:IDC and hypotype thereof

	>90％UCL	>95％UCL	>99％UCL	>99.9％UCL
					Normally	2.9％(2/68)	2.9％(2/68)	2.9％(2/68)	2.9％(2/68)
IDC	39.6％(67/169)	30.2％(51/169)	16.0％(27/169)	8.9％(15/169)
					IDC ER(+)	37.1％(13/35)	22.9％(8/35)	17.1％(6/35)	8.6％(3/35)
IDC ER(+)/PR(+)	38.5％(10/26)	23.1％(6/26)	154％(4/26)	7.7％(2/26)
					IDC ER(+)/PR(-)	37.5％(3/8)	25.0％(2/8)	25.0％(2/8)	12.5％(1/8)
IDC ER(-)	61.1％(11/18)	55.6％(10/18)	33.3％(6/18)	16.7％(3/18)
					IDC ER(-)/PR(-)	75.0％(6/8)	62.5％(5/8)	50.0％(4/8)	37.5％(3/8)
IDC Her2-neu(+)	50.0％(12/24)	29.2％(7/24)	25.0％(6/24)	12.5％(3/24)
					IDC Her2-neu(-)	80.0％(8/10)	70.0％(7/10)	40.0％(4/10)	30.0％(3/10)
IDC PR(+)	38.5％(10/26)	23.1％(6/26)	15.4％(4/26)	7.7％(2/26)
					IDC PR(-)	55.0％(11/20)	45.0％(9/20)	35.0％(7/20)	20.0％(4/20)
The IDC I phase	16.7％(3/18)	5.6％(1/18)	0.0％(0/18)	0.0％(0/18)
					The IDC II phase	44.3％(31/70)	35.7％(25/70)	12.9％(9/70)	4.3％(3/70)
The IDC III phase	42.9％(6/14)	35.7％(5/14)	214％(3/14)	14.3％(2/14)
					The IDC IV phase	20.0％(1/5)	20.0％(1/5)	0.0％(0/5)	0.0％(0/5)
IDC p53(+)	62.5％(5/8)	37.5％(3/8)	25.0％(2/8)	12.5％(1/8)
					IDC p53(-)	50.0％(8/16)	43.8％(7/16)	31.3％(5/16)	12.5％(2/16)

The result who is drawn by the form of summarizing is as follows:

Most IDC hypotype shows that at least 30% sample is higher than 95% UCL, has following significant exception:

Zero all IDC ER+ groups

○IDCHer2-neu+

Zero all IDC PR+ groups

Zero I phase and IV phase

The classification that PARP1 expresses compares:

○IDC ER->IDC ER+

○IDC Her2-neu->IDC Her2-neu+

○IDC PR->IDC PR+

○IDC p53-～＝IDC p53-

The II phase of zero IDC, III phase〉I phase, IV phase of IDC

The statistical study of (Curated) sample sets specially

Normal and IDC summarizes statistics

According to the information about sample, the expression in two normal specimens and the IDC sample is higher than that the reason of all the other samples is unconspicuous in the group.For ASCENTA ^TMThe regulation sample in the method for quality control carried out by Gene Logic comprise outlier evaluation to the multivariate level, but on array, use the full gene collection, and do not carry out concrete comparison with other sample sets.These samples were not determined to be in the outlier in the scope of the full gene collection of measuring on the HG-U133A array originally.For more closely assessing the sample in this specific set of data scope, we use one group of gene selecting in order to distinguish normal and infitrating ductal carcinoma to carry out quality evaluation.

Selection can be distinguished one group of about 1 700 gene of NBT and IDC, and carries out principal component analysis (PCA) and correlation analysis.The gene demonstration of each selection is at least 2 times of changes, and the p value of t-check is lower than 0.01.Analysis result shows that the sample of two outliers seems classification error, should remove.As the part of the research of two outliers in Fig. 4 a and Fig. 4 b, determining, the group of 237 samples is carried out bigger assessment.The result of these analyses shows that other 3 normal samples and 5 IDC samples should remove from this analysis.These samples seem classification error, and are not the samples that is suitable for this analysis.Remove the sample of 10 outliers, remaining 63 normal samples and 164 IDC samples.Following table E has described remaining sample number in each IDC subgroup in detail.

All subgroups continue to have at least 5 samples.As if an IDC sample that is defined as the outlier of PARP1 expression be not the outlier in this quality evaluation.This sample is retained in this analysis.

5 normal specimens of removing tend to be in the higher-end of normal expression scope.Thereby remove these 5 and tend to reduce population mean.In addition, remove 2 outliers especially and cause narrower cofidence limit.In the IDC classification, 5 outliers determining tend to be in IDC express scope than low side.Remove these samples and cause having improved slightly the summary statistics.Table F shows up-to-date summary statistics.Change in the IDC group is obvious unlike normal group, because sample number increases, also looks like the outlier of PARP1 because of neither one in 5 samples removing.

Table E: tumour number percent and pathological sample number (having removed outlier)

Group	25-50	50-75	75-90	>90	All
						Normally	N/A	N/A	N/A	N/A	63
IDC	14	36	59	55	164
						IDC ER(+)	9	9	11	5	34
IDCER(+)/PR(+)	7	7	8	3	25
						IDC ER(+)/PR(-)	1	2	3	2	8
IDC ER(-)	3	6	7	1	17
						IDC ER(-)/PR(-)			7	1	8
IDC Her2-neu(+)	8	5	10		23
						IDC Her2-neu(-)	1	3	4	1	9
IDC PR(+)	7	7	8	3	25
						IDC PR(-)	1	5	10	3	19
The IDC I phase		3	9	6	18
						The IDC II phase		19	21	28	68
The IDC III phase		2	8	4	14
						The IDC IV phase			2	3	5
IDC p53(+)	2	3	2		7
						IDC p53(-)	7	4	5		16

The removal of outlier sample causes the change multiple between IDC and normal mean intensity to increase.The t check of determining the significant difference between two groups causes the p value to reduce.In a word, the removal of outlier causes that bigger difference is arranged between the mean intensity of normal and IDC, and this species diversity is more remarkable.

Table F: the summary statistics that does not have the normal and IDC breast sample sets of outlier

Group	N	Mean value	Standard deviation	Standard error		90％UCL	95％UCL	99％UCL	99.9％UCL
										Infitrating ductal carcinoma	164	332.819	135.360	10.5698	557.421	600.918	686.686	787.821
Normal structure	63	186.413	40.367	5.0857	254.350	267.743	294.534	326.961

IDC mean value/normal mean value=1.79

T-check for (IDC mean value=normal mean value) produces p=2*10 ^-27

The single sample assessment

As observed in table C, when removing outlier, the confidence upper limit that calculates for normal specimens reduces.This causes more IDC sample outside the various scopes that limit.Fig. 5 a and Fig. 5 b have reflected the sample number of minimizing and the tighter cofidence limit of generation.

The result of comparison diagram 4a and Fig. 4 b, the mean value of normal group has dropped to and has been lower than 200, and confidence upper limit than whole 237 analyses more significantly near mean value.In various other tumour number percents of level, continuing does not have evident difference.This is based on following observation: be categorized as〉some samples of 90% tumour tend to be in the low side of infitrating ductal carcinoma scope, and the sample in the 25%-50% tumour classification has higher PARP1 expression.In addition, the type of 50%-75% and 75%-90% is tended to be uniformly distributed among the tumor sample expression scope.In a word, more IDC sample comparison analysis early is on each cofidence limit.

As observed in all samples is analyzed, the PARP1 in ER (-), PR (-) and Her2-neu (-) classification expresses and tends to a little more than its corresponding (+) classification.This discovery is not observed in p53 classification or each phase classification of tumour.Single sample has contribution for the multiple classifition in this analysis, and this fact can influence this conclusion.Observing supplementary data set, to disclose the highest PARP1 expresser in ER (-) group be identical high expressed person in PR (-) and Her2-neu (-) organize.For the minimum expression in (+) group equally also is like this.

As in this part than what predict previously, the IDC sample number that is higher than normal UCL increases along with removing outlier.The sample number that table G has summarized each cofidence limit that is higher than various types of other infitrating ductal carcinoma.For making 164 as a whole IDC samples, to compare with 9% with former 39%, 74% and 45% sample is higher than 90% and 99.9% UCL respectively.(-) status categories its corresponding (+) classification of ER, PR and Her2-neu is compared and is still kept improving.When respectively organizing on the UCL level 99.9%, difference is the most obvious.Compare with the Her2-neu group with ER, the difference in the PR classification is more not obvious.

Table G: the number percent and the quantity of removing the sample of the IDC of outlier and the UCL that hypotype surpasses thereof

	>90％UCL	>95％UCL	>99％UCL	>99.9％UCL
					Normally	7.9％(5/63)	4.8％(3/63)	1.6％(1/63)	0.0％(0/63)
IDC	744％(122/164)	70.1％(115/164)	58.5％(96/164)	45.7％(75/164)
					IDC ER(+)	73.5％(25/34)	73.5％(25/34)	61.8％(21/34)	38.2％(13/34)
IDC ER(+)/PR(+)	72.0％(18/25)	72.0％(18/25)	60.0％(15/25)	40.0％(10/25)
					IDC ER(+)/PR(-)	75.0％(6/8)	75.0％(6/8)	62.5％(5/8)	37.5％(3/8)
IDC ER(-)	88.2％(15/17)	88.2％(15/17)	76.5％(13/17)	64.7％(11/17)
					IDC ER(-)/PR(-)	75.0％(6/8)	75.0％(6/8)	75.0％(6/8)	75.0％(6/8)

	>90％UCL	>95％UCL	>99％UCL	>99.9％UCL
					IDC Her2-neu(+)	82.6％(19/23)	82.6％(19/23)	73.9％(17/23)	52.2％(12/23)
IDC Her2-neu(-)	88.9％(8/9)	88.9％(8/9)	88.9％(8/9)	88.9％(8/9)
					IDC PR(+)	72.0％(18/25)	72.0％(18/25)	60.0％(15/25)	40.0％(10/25)
IDC PR(-)	78.9％(15/19)	789％(15/19)	73.7％(14/19)	57.9％(11/19)
					The IDC I phase	50.0％(9/18)	444％(8/18)	33.3％(6/18)	22.2％(4/18)
The IDC II phase	75.0％(51/68)	69.1％(47/68)	60.3％(41/68)	50.0％(34/68)
					The IDC III phase	71.4％(10/14)	71.4％(10/14)	57.1％(8/14)	50.0％(7/14)
The IDC IV phase	80.0％(4/5)	60.0％(3/5)	20.0％(1/5)	20.0％(1/5)
					IDC p53(+)	85.7％(6/7)	85.7％(6/7)	85.7％(6/7)	71.4％(5/7)
IDC p53(-)	81.3％(13/16)	81.3％(13/16)	75.0％(12/16)	56.3％(9/16)

Conclusion:

Compare normal specimens, the expression of the PARP1 in infitrating ductal carcinoma obviously improves.Although Fig. 5 a and Fig. 5 b show these discoveries being arranged, is not all overexpressions of IDC sample.The distribution of this broad and towards the more displacement of high expressed in the IDC group shows that the PARP1 that about 70% IDC can have above 95% confidence upper limit of normal population expresses.The viewed discovery of former BiPar has been supported in this discovery.Further analysis to each subgroup of IDC sample discloses, if their ER state is negative, or their Her2-neu state is negative, and observed PARP1 expresses the number percent of the IDC that improves and brings up to 88%-89%.The number percent that is higher than the PR negative sample of normal 95%UCL is 79%, and is more not obvious, but still raise.

This shows, tests in the negative situation at ER, PR or Her2-neu, and any therapy at the PARP1 overexpression all is more effective.

In a word:

1. in infitrating ductal carcinoma than in NBT, the expression of PARP1 is higher.

2. as if the number percent of the tumour of observing in histopathologic slide be not to measure the key factor that PARP1 expresses.

3. the existence of an outlier may show in the individuality at little number percent and has unusual high expression in IDC group.

4. as if some hypotype of infitrating ductal carcinoma demonstrates the expression higher than other hypotype.Especially, (-) hypotype its corresponding (+) hypotype of ER, Her2-neu and PR is compared, and shows the higher sample number percent that is higher than normal UCL.

Discuss and explanation

The result of this research raises consistent with the expression of the PARP1 in infiltration ductal carcinomas of breast.If the overexpression of the PARP1 among the IDC is defined as level greater than 95% confidence upper limit of expressing in the NBT, so about 2/3 infitrating ductal carcinoma overexpression PARP1.If the overexpression of PARP1 defines the response of the raising that suppresses for PARP1, the result means that most IDC is the rational candidate with the treatment of PARP1 inhibitor so.In addition, in the IDC subgroup of estrogen receptor negative and Her2-neu feminine gender, the ratio of the tumour of overexpression PARP1 even than higher in the entire I DC colony shows: (1) uses the standard laboratory analysis to be absorbed in the particular type of tumour of PARP1 overexpression in clinical testing or the difference response of estimating for treatment is favourable; (2) the PARP1 inhibition is a kind of reasonable method for the cancer that can not use antiestrogenic and anti-Her2-neu treatment.

Embodiment 3

The tissue expression of IARP1 in oophoroma and normal ovarian

Research and design

Normal ovarian sample and carcinous ovary sample are selected from System, System be for

The member of the sample sets of system's regulation.Be noted that any carcinous sample can occur in more than a hypotype grouping.In table H, the ovary sample of 10 selections and an example of the membership qualification in many hypotypes thereof have been shown.For example, sample GID8757 is classified as endometrium template cancer, with and corresponding age, CA125 state and hypotype by stages.Some hypotype is got rid of each other, and other hypotype then is not like this, produces the complete categorizing system for any single sample.

Table H: for the example of the hypotype classification of the ovary sample of selecting

The genomics sign	Normal ovarian	Hyaline cell	Endometrial-like	Endometrial-like was above 45 years old	Endometrial-like is lower than 45 years old	Endometrial-like, CA125 raises	Endometrial-like, the I phase	Endometrial-like, the III phase
									4051		Y
9357		Y
									7473		Y

31852		Y
									15133		Y
12007		Y
									7389			Y	Y
8757			Y	Y		Y	Y
									2619			Y	Y			Y
31903			Y	Y		Y	Y

On the HG-U133A array, be that the single probe sets of " W208644_at " is represented the PARP1 gene by identifier.All results in this report produce based on the MAS5 expression signal intensity for this probe sets, and are called ＂ PARP1 ＂.

Statistical study

The summary statistics of normal and cancer

Normal and main cancer sample classification is pressed mean value, standard deviation, standard error and some confidence upper limit summaries based on the t distribution.Confidence upper limit (UCL) is similar to the standard deviation statistics, because they determine the specific region of the probability of a value of observation.For example, 95% confidence upper limit is similar to the value of predicting in this will have an opportunity sample 5% more than value.

For the ovary normal data, sample number (n=88) is enough big, makes t distribute and extremely be similar to the result who obtains when only using standard deviation to set the limit of summarizing as Table I.For example, the mean value of normal ovarian expression intensity+2 standard deviations are 224.18, and it is very similar to 95% cofidence limit 224.15.The organ that the normal specimens number is lower then is not like this.

Table I: the summary statistics of normal and carcinous ovary sample sets

Main carcinous sample classification	Quantity	Mean value	Standard deviation	Standard error		90％UCL	95％UCL	99％UCL	99.9%UCL
										Normal structure	88	163.037	30.572	3.259	214.15	224.15	244.00	267.75
Clear cell adenocarcinoma	6	220.757	45.995	18.777	320.86	348.46	421.07	562.00
									Endometrioid adenocarcinoma	13	302.863	119.713	33.202	524.28	573.54	682.33	839.27
GCT	3	422.980	204.006	117.783	1110.83	1436.54	2760.94	7866.65
									MCAC	7	191.453	47.990	18.139	291.14	316.99	381.66	497.16
Seedling is reined in mixed tumour	5	371404	144.270	64.520	708.32	810.19	1099.04	1732.18
									Papillary serous adenocarcinoma	64	357.092	144.994	18.124	601.03	649.09	745.21	861.47
Serous cystadenocarcinoma	8	371.234	104.078	36.797	580.38	632.27	757.55	968.22

All oophoromas are expressed higher mean P ARP1 than normal ovarian.Clear cell adenocarcinoma and MCAC sample be than the significantly lower PARP1 of other subtype expression, and the variation of expressing also is lower than, and Fig. 6 shows.

Table J listed PARP1 gene that the array data that uses Table I records based on the change multiple of ratio and the result of the two tail t checks of Student.

Table J: cancer types and normal comparative statistics data

Change multiple very greatly although be noted that some, the small sample capacity can produce insignificant p value, as observed for GCT.In addition, papillary serous adenocarcinoma contains a large amount of samples, and produces the p value of highly significant, is lower than the observed ratio change of GCT group even its ratio changes.Need limit the size of estimating effect and based on the conspicuousness of variance, to explain its result in conjunction with sample capacity.

The single sample assessment

Secondly, with respect to normal ovary sample distribution, test the single sample of all oophoroma hypotypes separately.Each is defined as 90%, 95%, 99% and 99.9% confidence upper limit above normal group.The carcinous ovary sample of neither one is lower than 111.92 90% confidence lower limit, so do not propose the LCL boundary.

Fig. 6 shows the result's of each ovary sample classification visual summary.Each symbology basis is at the disease type of x-axle demonstration and the simple sample of drawing at the PARP1 expression intensity that the y-axle shows.Draw the reference line of the normal UCL of expression 90%, 95%, 99% and 99.9% with horizontal dotted line.Mean value with horizontal reference depicted as solid lines normal specimens.

Can make some following explanations according to Fig. 6:

Compare normal ovary sample, in carcinous ovary sample, the rising that PARP1 expresses is tangible.

PARP1 in the PARP1 expression ratio normal ovarian sample in the carcinous ovary sample expresses the variation that shows much higher degree.

Express about PARP1, in normal ovary sample sets, do not observe outlier.

Table K has summarized number percent and the quantity that surpasses for the sample of the predefined confidence upper limit of oophoroma classification.

Table K: the number percent and the quantity of the sample that surpasses UCL of oophoroma hypotype

Form by general introduction draws the following result:

Most of pathology hypotype of oophoroma shows that most of sample is higher than 95% UCL:

Palilate slurry type, serous cystadenocarcinoma, GCT and Miao Le mixed tumour all have the similar high sample incidence that is higher than 95% UCL.

In uterus in the film sample gland cancer, only about half of sample is higher than 95% UCL.

In clear cell adenocarcinoma and MCAC, 1/3 or sample still less be higher than 95% UCL.

The clinical subtype that PARP1 expresses relatively discloses:

The palilate slurry type I phase is similar to the palilate slurry type III phase.

The palilate slurry type that CA125 raises is similar to the palilate slurry type.

The comparison of PARP1 and selected gene

The expression of PARP1 with as other expression of gene of concentrate measuring at the HG-U133A/B array relevant.The relevant whole set that are based upon 194 samples of this analysis selection.Table L has summarized the result of this analysis.

The expression of table L:PARP1 is relevant with the Pearson of selected probe sets

Gene symbol	Fragment	Correlativity with 208644_at (PARP1)
			BRCA1	204531_s_at	0.314
BRCA2	214727at	0.274

Positive correlation shows that probe sets changes along the direction identical with PARP1.When PARP1 has low the expression, for example, in normal specimens, expect that these Expression of Related Genes are also lower.When PARP1 has the expression of raising, for example, in pernicious sample, expect that these Expression of Related Genes also improve.

Conclusion

Compare normal specimens, the expression of the PARP1 in the oophoroma sample improves.Although Fig. 6 shows this discovery is arranged, I met not is that all oophoroma samples all show this overexpression.The distribution of this broad and towards the more displacement of high expressed in the oophoroma group shows that the PARP1 that about 75% oophoroma has 95% confidence upper limit of expressing above normal ovarian expresses.Further analysis to each subgroup of oophoroma sample discloses, and reins in mixed tumour or GCT hypotype if they are papillary serous adenocarcinoma, serous cystadenocarcinoma, seedling, observes the number percent that PARP1 expresses the oophoroma sample that raises and brings up to about 90%.In 1/3 or still less the sample of estimating, clear cell adenocarcinoma and serous cystadenocarcinoma do not show the PARP1 of raising.

In a word:

1. in oophoroma than in normal ovarian tissue, the expression of PARP1 is higher.

2. as if some hypotype of oophoroma demonstrates the expression higher than other hypotype.Especially, papillary serous adenocarcinoma, serous cystadenocarcinoma, seedling are reined in mixed tumour and GCT sample, the sample number percent that is higher than normal UCL that demonstration is higher than endometrial-like, endometrial-like show again than clear cell adenocarcinoma and the higher sample number percent that is higher than normal UCL of MCAC.

Discuss and explanation

If the overexpression of the PARP1 in the oophoroma is defined as level greater than 95% confidence upper limit of expressing in the normal ovarian tissue, so about 75% oophoroma sample overexpression PARP1.If the overexpression of PARP1 defines the response of the raising that suppresses for PARP1, this result means that most oophoroma is the rational candidate of PARP1 inhibitor for treating so, especially reins in mixed tumour and GCT for papillary serous adenocarcinoma, serous cystadenocarcinoma, seedling.

Embodiment 4

The gene expression of PARP1 in pernicious and normal uterus inner membrance, lung and prostata tissue sample

This plan is by study the expression of PARP1mRNA in people's normal uterus inner membrance (n=23), lung (n=122) and prostate (n=57) and various carcinomas of endometrium (n=57), lung cancer (n=101) and prostate cancer (n=57) in mensuration on the Affymetrix HG-U133A/B array collection.

The PARP1 that the main target of this research is based in the normal structure sample expresses, by using objective statistics threshold value, and " overexpression " of definition PARP1mRNA, and determine and characterize the cancer sample that surpasses this statistics threshold value.

Compare with normal specimens, the PARP1 in the cancer expresses general the rising.About 3/4 and all prostate cancer samples of about 1/4, all lung cancer samples of all carcinoma of endometrium samples about 1/8 in, the expression of PARP1 surpasses 95% confidence upper limit (" overexpression ") of normal population.Seedling reins in mixed tumour and squamous cell lung carcinoma shows that the highest PARP1 expresses the incidence that raises.The expression of PARP1 in adenocarcinoma of the prostate is starkly lower than the cancer of estimating in the in uterus membrane tissue and lung tissue.

The correlation research of other gene of PARP1 and all has determined that the gene relevant with PARP1 is up to 80%.In uterus in film and the lung sample, determine that the one group common gene relevant with cell proliferation height (that is, be in 40 high value) in two kinds of tissues is correlated with.

This plan of analysis is by studying the expression of PARP1mRNA in the normal and carcinous endometrium of people, lung and prostate sample in mensuration on the Affymetrix HG-U133A/B array collection.This analysis has proposed following target:

-with from same or medically similarly the control sample (that is, " normally ") of types of organization compare, characterize the imitate PARP1 expression of product of single endometrium, lung and tumor of prostate.

-sign is expressed with respect to the PARP1 of all other gene expressions on HG-U133A/B array collection.

Research and design

Selection is from the single normal and carcinous sample of endometrium, lung and prostata tissue.Any carcinous sample can occur in more than a kind of hypotype grouping.In table M, the endometrium sample of 10 selections and an example of the membership qualification in multiple hypotype thereof have been shown.

Table M: the example of the hypotype classification of the endometrium sample of selection

GID	Normally	Pernicious	Gland cancer, endometrial-like	Gland cancer, endometrial-like, no smoking history	Gland cancer, endometrial-like, obesity	Gland cancer, endometrial-like, obesity, no smoking history	Gland cancer, endometrial-like is after the menopause	Gland cancer, endometrial-like is before the menopause	Gland cancer, endometrial-like, I phase	Gland cancer, endometrial-like, non-obesity	Seedling is reined in mixed tumour	Gland cancer, endometrial-like, obesity, smoking history
													565	Y
612	Y
													1109	Y
1119	Y
													1146		Y	Y	Y			Y
1427		Y	Y	Y				Y
													1638	Y
1815	Y
													2401		Y	Y	Y					Y
2402		Y	Y	Y			Y		Y	Y

On the HG-U133A array, be that single probe sets of " W208644_at " is represented the PARP1 gene by identifier.All results produce based on the MAS5 expression signal intensity for this probe groups, and are called ＂ PARP1 ＂.

Statistical study-endometrium result

Normal and pernicious sample classification is pressed mean value, standard deviation, standard error and some confidence upper limit summaries based on the t distribution.Confidence upper limit (UCL) is similar to the standard deviation statistics, because they determine the specific region for the probability of observing a value.For example, 95% confidence upper limit is similar to the value predicted of will having an opportunity in 5% sample on this value.

Table N shows the summary statistics for each normal and carcinous endometrium sample collection.

Table N: the summary statistics of normal and carcinous endometrium sample collection

Group	Quantity	Mean value	Standard deviation	Standard error		90％UCL	95％UCL	99％UCL	99.9％UCL
										Normally	23	201.21	62.21	12.97	310.33	333.00	380.34	442.20
Gland cancer, endometrial-like	50	297.42	98.78	13.97	464.67	497.89	564.77	646.62
									Gland cancer, endometrium,	40	286.55	91.55	14.47	442.71	474.02	537.53	616.33

No smoking history
									Gland cancer, endometrial-like, non-obesity	3	373.40	76.85	44.37	632.53	755.23	1254.16	3177.60
Gland cancer, endometrial-like, obesity	15	291.22	79.84	20.61	436.46	468.08	536.69	632.64
									Gland cancer, endometrial-like, obesity, no smoking history	9	260.84	7344	24.48	404.79	439.35	520.58	651.08
Gland cancer, endometrial-like, obesity, smoking history	6	336.79	71.19	29.06	491.73	53444	646.82	864.94
									Gland cancer, endometrial-like is after the menopause	35	308.83	97.96	16.56	476.82	510.73	579.90	666.56
Gland cancer, endometrial-like is before the menopause	3	250.09	12.48	7.20	292.15	312.07	393.07	705.32
									Gland cancer, endometrial-like, smoking history	6	336.79	71.19	29.06	491.73	53444	646.82	864.94
Gland cancer, endometrial-like, I phase	9	340.80	68.13	22.71	474.34	506.40	581.76	702.84
									Seedling is reined in mixed tumour	7	517.86	185.55	70.13	903.31	1003.23	1253.26	1699.84

The mean P ARP1 signal intensity that all carcinomas of endometrium are higher than film expression in the normal uterus.Seedling is reined in the mixed tumour sample than the obvious higher PARP1 of other subtype expression.Among this Fig. 7 below as can be seen.

The result that the change multiple and the two tail t of Student based on ratio of PARP1 gene expression checked when table O had listed with normal specimens relatively.

Table O: the comparative statistics data of endometrioid carcinoma type and normal uterus inner membrance

The cancer group	Change multiple (cancer/normal)	T check p value (with respect to normal)
			Gland cancer, endometrial-like	1.48	3.972E-06
Gland cancer, endometrial-like, no smoking history	1.42	4.740E-05

Gland cancer, endometrial-like, non-obesity	1.86	5.035E-02
			Gland cancer, endometrial-like, obesity	145	1.088E-03
Gland cancer, endometrial-like, obesity, no smoking history	1.30	5.109E-02
			Gland cancer, endometrial-like, obesity, smoking history	1.67	3.596E-03
Gland cancer, endometrial-like is after the menopause	1.53	3.947E-06
			Gland cancer, endometrial-like is before the menopause	1.24	3.941E-03
Gland cancer, endometrial-like, smoking history	1.67	3.596E-03
			Gland cancer, endometrial-like, I phase	1.69	1.172E-04
Seedling is reined in mixed tumour	2.57	3.721E-03

Then, distribute, detect the single sample of all carcinoma of endometrium hypotypes separately with respect to normal endometrium sample.Each is defined as surpassing 90%, 95%, 99% and 99.9% confidence upper limit of normal group.

Fig. 7 shows each other result's of endometrial-like category visual summary.Each symbology basis is at the disease type of x-axle demonstration and the single sample of drawing at the PARP1 expression intensity that the y-axle shows.Draw the reference line of the normal UCL of expression 90%, 95%, 99% and 99.9% with horizontal dotted line.Mean value with horizontal reference depicted as solid lines normal specimens.

With respect to normal endometrium sample, in carcinous endometrium sample, it is tangible that the PARP1 of raising expresses.PARP1 in the PARP1 expression ratio normal uterus in the carcinous endometrium sample in the membrane sample expresses the variation that shows much higher degree (that is, bigger diffusion).About the expression of PARP1, in normal endometrium sample, do not observe outlier.

Table P has summarized number percent and the quantity that surpasses for the sample of the predefined confidence upper limit of carcinoma of endometrium type.This table has surpassed the classification of type of 90%UCL by the maximum sample incidence.Therefore, the type on close form top contains the most a high proportion of sample above normality threshold.

Table P: the number percent (counting) of the sample that surpasses UCL of carcinoma of endometrium hypotype

	90％UCL	95％UCL	99％UCL	99.9％UCL
					Normally	4.3％(1/23)	4.3％(1/23)	4.3％(1/23)	0.0％(0/23)
Gland cancer, endometrial-like, non-obesity	100.00％(3/3)	33.3％(1/3)	33.3％(1/3)	33.3％(1/3)
					Seedling is reined in mixed tumour	85.7％(6/7)	85.7％(6/7)	714％(5/7)	71.4％(5/7)
Gland cancer, endometrial-like, obesity, smoking history	83.3％(5/6)	50.0％(3/6)	16.7％(1/6)	0.0％(0/6)
					Gland cancer, endometrial-like, smoking history	83.3％(5/6)	50.0％(3/6)	16.7％(1/6)	0.0％(0/6)
Gland cancer, endometrial-like, I phase	66.7％(6/9)	33.3％(3/9)	22.2％(2/9)	11.1％(1/9)
					Gland cancer, endometrial-like,	53.3％(8/15)	26.7％(4/15)	6.7％(1/15)	0.0％(0/15)

Fat
					Gland cancer, endometrial-like is after the menopause	514％(18/35)	37.1％(13/35)	20.0％(7/35)	11.4％(4/35)
Gland cancer, endometrial-like	46.0％(23/50)	30.0％(15/50)	18.0％(9/50)	10.0％(5/50)
					Gland cancer, endometrial-like does not have smoking history	40.0％(16/40)	25.0％(10/40)	15.0％(6/40)	7.5％(3/40)
Gland cancer, endometrial-like, obesity, no smoking history	33.3％(3/9)	11.1％(1/9)	0.0％(0/9)	0.0％(0/9)
					Gland cancer, endometrial-like is before the menopause	0.0％(0/3)	0.0％(0/3)	0.0％(0/3)	0.0％(0/3)

Most of pathology hypotype of carcinoma of endometrium shows that most of samples surpass 90% UCL.What pay special attention to is that seedling is reined in mixed tumour and had the maximum sample incidence (85.7%) that surpasses 95% UCL, and keeps high rate (71.4%) when 99.9% UCL.

The result of lung

Normal and pernicious sample type is pressed mean value, standard deviation, standard error and some confidence upper limit summaries based on the t distribution.Confidence upper limit (UCL) is similar to the standard deviation statistical value, because they determine the specific region of the probability of a value of observation.For example, 95% confidence upper limit is similar to the value that will have an opportunity in the prediction of 5% sample on this value.

Table Q shows the summary statistics for each normal and carcinous lung sample sets.

Table Q: the summary statistics of normal and carcinous lung sample sets

Group	Quantity	Mean value	Standard deviation	Standard error		90％UCL	95％UCL	99％UCL	99.9％UCL
										Normally	122	162.37	32.85	2.97	217.03	227.66	248.68	273.60
The gland carcinoma squamosum	3	209.41	25.20	14.55	294.36	334.59	498.17	1128.78
									Gland cancer	46	284.99	92.24	13.60	441.58	472.79	535.77	613.23
Gland cancer, smoking history	27	276.68	54.55	10.50	371.43	390.86	431.03	482.57
									Gland cancer, the I phase	10	244.47	43.66	13.81	328.41	348.06	393.29	463.40
Gland cancer, the II phase	7	301.52	64.51	24.38	435.53	470.27	557.19	712.45
									Gland cancer, the III phase	5	301.58	85.87	3840	502.11	562.74	734.66	111149
Large cell carcinoma	7	291.08	122.74	46.39	546.06	612.16	777.56	1072.98
									Large cell carcinoma, smoking history	6	256.71	90.31	36.87	453.27	507.46	650.03	926.74
Large cell carcinoma, the I phase	4	356.73	110.50	55.25	647.46	749.89	1078.32	1953.37
									Neuroendocrine carcinoma (non-small cell)	3	408.91	287.69	166.10	1378.91	1838.22	3705.88	10905.91
Small cell carcinoma	3	473.23	239.88	138.49	1282.03	1665.02	3222.30	9225.83

Small cell carcinoma, the II phase	3	473.23	239.88	138.49	1282.03	1665.02	3222.30	9225.83
									Squamous cell carcinoma	39	309.53	103.71	16.61	486.62	522.16	594.34	684.05
Squamous cell carcinoma, smoking history	36	310.91	107.51	17.92	495.06	532.17	607.78	702.31
									Squamous cell carcinoma, the I phase	16	315.57	78.05	19.51	456.60	487.04	552.63	643.22
Squamous cell carcinoma, the II phase	5	291.67	30.10	13.46	361.98	383.23	443.50	575.61
									Squamous cell carcinoma, the III phase	5	236.10	63.69	2848	384.83	429.80	557.30	836.79

All lung cancer are expressed higher mean P ARP1 signal intensity than normal lung.Among this Fig. 8 below as can be seen.

The result that the change multiple and the two tail t of Student based on ratio of PARP1 gene expression checked when table R had listed with normal specimens relatively.

Table R: the comparative statistics data of lung cancer type and normal lung

The cancer group	Change multiple (cancer/normal)	T check p value (with respect to normal)
			The gland carcinoma squamosum	1.29	7.811 E-02
Gland cancer	1.76	1.073 E-11
			Gland cancer, smoking history	1.70	1.359 E-11
Gland cancer, the I phase	1.51	1.800 E-04
			Gland cancer, the II phase	1.86	1.176 E-03
Gland cancer, the III phase	1.86	2.201 E-02
			Large cell carcinoma	1.79	3.220 E-02
Large cell carcinoma, smoking history	1.58	5.062 E-02
			Large cell carcinoma, the I phase	2.20	3.876 E-02
Neuroendocrine carcinoma (non-small cell)	2.52	2.760 E-01
			Small cell carcinoma	2.91	1.539 E-01
Small cell carcinoma, the II phase	2.91	1.539 E-01
			Squamous cell carcinoma	1.91	7.722 E-11
Squamous cell carcinoma, smoking history	1.91	8.215 E-10
			Squamous cell carcinoma, the I phase	1.94	9.249 E-07
Squamous cell carcinoma, the II phase	1.80	4.516 E-04
			Squamous cell carcinoma, the III phase	1.45	6.037 E-02

Then, with respect to the normal lung sample distribution, detect the single sample of all lung cancer hypotypes separately.Each is defined as surpassing 90%, 95%, 99% and 99.9% confidence upper limit of normal group.The carcinous lung sample of neither one is lower than 90% confidence lower limit of normal specimens, so do not propose the LCL boundary.

Fig. 8 shows the result's of each lung sample type visual summary.Each symbology basis is at the disease type of x-axle demonstration and the single sample of drawing at the PARP1 expression intensity that the y-axle shows.Draw the reference line of the normal UCL of expression 90%, 95%, 99% and 99.9% with horizontal dotted line.Mean value with horizontal reference depicted as solid lines normal specimens.With respect to normal lung sample, in carcinous lung sample, it is tangible that the PARP1 of raising expresses.PARP1 in the expression ratio normal uterus of PARP1 in the carcinous lung sample in the membrane sample expresses the variation that shows much higher degree (that is, bigger diffusion).

Table S has summarized number percent and the quantity that surpasses for the sample of the predefined confidence upper limit of lung cancer type.This table has surpassed the classification of type of 90%UCL according to the maximum sample incidence.Therefore, the type near the form top contains the most a high proportion of sample that surpasses normality threshold.

Table S: the number percent (counting) of the sample that surpasses UCL of lung cancer hypotype

	>90％UCL	>95％UCL	>99％UCL	>99.9％UCL
					Normally	4.9％(6/122)	3.3％(4/122)	1.6％(2/122)	0.0％(0/122)
Small cell carcinoma	100.00％(3/3)	100.00％(3/3)	100.00％(3/3)	100.00％(3/3)
					Small cell carcinoma, the II phase	100.00％(3/3)	100.00％(3/3)	100.00％(3/3)	100.00％(3/3)
Large cell carcinoma, the I phase	100.00％(4/4)	100.00％(4/4)	100.00％(4/4)	75.00％(3/4)
					Squamous cell carcinoma, the II phase	100.00％(5/5)	100.00％(5/5)	100.00％(5/5)	60.00％(3/5)
Neuroendocrine carcinoma (non-small cell)	100.00％(3/3)	100.00％(3/3)	66.7％(2/3)	33.3％(1/3)
					Squamous cell carcinoma, the I phase	87.5％(14/16)	87.5％(14/16)	81.3％(13/16)	66.8％(11/16)
Squamous cell carcinoma	87.2％(34/39)	82.1％(32/39)	744％(29/39)	61.5％(24/39)
					Squamous cell carcinoma, smoking history	86.1％(31/36)	80.6％(29/36)	75.0％(27/36)	61.1％(22/36)
Gland cancer, the II phase	85.7％(6/7)	85.7％(6/7)	85.7％(6/7)	57.1％(4/7)
					Gland cancer, smoking history	85.2％(23/27)	85.2％(23/27)	74.1％(20/27)	40.7％(11/27)
Gland cancer, the III phase	80.0％(4/5)	80.0％(4/5)	80.0％(4/5)	80.0％(4/5)
					Squamous cell carcinoma, the III phase	80.0％(4/5)	60.0％(3/5)	20.0％(1/5)	20.0％(1/5)
Gland cancer	76.1％(35/46)	73.9％(34/46)	63.0％(29/46)	37.0％(17/46)
					Large cell carcinoma	71.4％(5/7)	71.4％(5/7)	71.4％(5/7)	57.1％(4/7)
Gland cancer, the I phase	70.0％(7/10)	70.0％(7/10)	60.0％(6/10)	20.0％(2/10)
					Large cell carcinoma, smoking history	66.7％(4/6)	66.7％(4/6)	66.7％(4/6)	50.0％(3/6)
The gland carcinoma squamosum	33.1％(1/3)	33.1％(1/3)	0.0％(0/3)	0.0％(0/3)

The prostate result

Table T shows the summary statistics for each normal and carcinous prostate sample sets.

Table T: the summary statistics of normal and carcinous prostate sample sets

Group	Quantity	Mean value	Standard deviation	Standard error		90％UCL	95％UCL	99％UCL	99.9％UCL
										Normally	57	209.09	36.61	4.85	270.86	283.08	307.57	337.36
Gland cancer, 60 years old and more than	57	237.80	40.49	5.36	306.11	319.61	346.70	379.63

The prostate cancer group is expressed slightly high mean P ARP1 signal intensity than normal prostatic group.This in Fig. 9 as can be seen.

The result that the change multiple and the two tail t of Student based on ratio of PARP1 gene expression checked when table U had listed with normal specimens relatively.

Table U: the comparative statistics data of prostate cancer type and normal prostatic

The cancer group	Change multiple (cancer/normal)	T check p value (with respect to normal)
			Gland cancer, 60 years old and more than	1.14	1.273E-04

Fig. 9 shows the result's of each prostate sample type visual summary.Each symbology basis is at the disease type of x-axle demonstration and the single sample of drawing at the PARP1 expression intensity that the y-axle shows.Draw the reference line of the normal UCL of expression 90%, 95%, 99% and 99.9% with horizontal dotted line.Mean value with horizontal reference depicted as solid lines normal specimens.With respect to normal prostate sample, in carcinous prostate sample, the expression of the PARP1 of Ti Gaoing is tangible slightly.PARP1 in the carcinous prostate sample express show with the normal prostatic sample in PARP1 express degree similarly make a variation (that is the diffusion of Xiang Denging).

Table V has been summarized number percent and the quantity that surpasses for the sample of the predefined confidence upper limit of prostate cancer type.

Table V: the number percent (counting) of the sample that surpasses UCL of prostate cancer hypotype

	>90％UCL	>95％UCL	>99％UCL	>99.9％UCL
					Normally	7.0％(4/57)	1.8％(1/57)	0.0％(0/57)	0.0％(0/57)
Gland cancer, 60 years old and more than	17.5％(10/57)	12.3％(7/57)	7.0％(4/57)	0.0％(0/57)

In surpassing the slightly high sample incidence of 90%, 95% and 99% UCL threshold value, reflected once more 60 years old and the above high PARP1 expression of adenocarcinoma of the prostate part omitted.The all samples of normal group and carcinous group is all within 99.9% UCL scope.

These results mean that the carcinoma of endometrium of most lung cancer and selection is that rational candidate, the especially seedling of PARP1 inhibitor for treating reined in mixed tumour and squamous cell lung carcinoma.Expression in PARP1 Endometrial Carcinomas and the lung cancer is higher than the expression in its normal structure separately.As if the carcinoma of endometrium of some hypotype and lung cancer show higher expression than other hypotype.Specifically, seedling is reined in mixed tumour and the squamous cell lung carcinoma sample demonstration sample number percent that is higher than normal UCL higher than other type.

Embodiment 5

The expression of monitoring PARP in tissue sample

Analyze and describe and method

XP ^TM-PCR is multiple (multiplex) RT-PCR method (Quin-Rong Chen of a kind of permission expression of analyzing several genes in a reaction, Gordon Vansant, KahukuOades, Maria Pickering, Jun S.Wei, Young K.Song, Joseph Monforte, with Javed Khan:Diagnosis of the Small Round Blue Cell Tumors UsingMutliplex Polymerase Chain Reaction.Journal of Molecular Diagnostics, Vol.9.No.1, in February, 2007).The combination of the gene-specific primer that uses in reaction and the qualification of universal primer produces a series of fluorescently-labeled PCR products, uses capillary electrophoresis GeXP to measure the size and the content of this PCR product.

Sample preparation

Briefly, with the tissue sample of fresh purifying with 6 X 10 ⁶Cells/well is coated with and is laid in 24 orifice plates.Half sample dissolves immediately, and remaining is snap frozen in dry ice and ethanol bath, and preserves 24 hours at-80 ℃.According to Althea Technologies, total RNA partition method of Inc.SOP is used the total RNA in the every duplicate samples of Promega SV96 kit (Cat.No.Z3505) separation.Use Quant-it Ribogreen RNA to measure kit (Cat.No.R-11490) by 03-XP-008, RNA quantitatively obtains the concentration of the RNA that obtains from every duplicate samples.Adjustment is carried out XP then from a part of RNA to 5ng/ μ L of every duplicate samples ^TM-PCR.

XP ^TM-PCR

Scheme (the Quin-Rong Chen that describes before using, Gordon Vansant, KahukuOades, Maria Pickering, Jun S.Wei, Young K.Song, Joseph Monforte, with Javed Khan:Diagnosis of the Small Round Blue Cell Tumors UsingMutliplex Polymerase Chain Reaction.Journal of Molecular Diagnostics, Vol.9.No.1, in February, 2007), use the total RNA of 25ng of every duplicate samples to carry out multiple RT-PCR.Press SOP11-XP-002, the cDNA Production from RNA with the AppliedBiosystems 9700 described RT that carry out react.In order to monitor the efficient of RT and PCR reaction, 0.24 vast mole kanamycins RNA is joined in each RT reaction.Use two types positive control RNA.Other analysis of control comprise ' no template contrast ' (NTC) and ' subtracting reverse transcriptase ' (RT-) contrast, among the former, add entry but not RNA separately reacts; Among the latter, sample RNA does not have the program of reverse transcriptase.

Expression analysis and calculating

Analyze the PCR reaction by Capillary Electrophoresis.With the dilution of fluorescently-labeled PCR reactant, with genome laboratory size criteria-400 (Beckman-Coulter, Part Number 608098) mixing, sex change and utilize SOP 11-XP-004, Operation and Maintenance of the CEQ8800 Genetic Analysis System application of sample is to Beckman Coulter.From 8800 data that obtain, produce relative expression's value of each gene with the expression analysis software analysis.The expression that each target gene is expressed with respect to the cyclophilin A in the same reaction (cyclophilin A), GAPDH or beta-actin is reported with the mean value of replication.Under the suitable situation, also report is worth the relevant standard deviation and the number percent coefficient of variation (%CV) with these.

Statistical analysis technique

The mathematical form that is used for the ANOVA model of this analysis is:

Wherein, Y _Ijk1Be k the time point that duplicates since the 1st time, under j dose concentration, the standardized Rfu ratio that in i sample, obtains.Model parameter μ is total standardized Rfu ratio of mean value, the constant an of the unknown, α _iBe the fixed effect that sample i causes, β _jBe the fixed effect that dose concentration j causes, γ _kBe the fixed effect that time point k causes, ω _{1 (ijk)}Be under j dose concentration, at k time point, duplicate the random effect that causes the 1st time in the 1st sample, suppose that it is normal distribution, mean value is 0, and variance is σ ² _ωε _Ijk1Be and k the time point that duplicates since the 1st time, under j dose concentration, by the relevant stochastic error item of standardized Rfu ratio that i sample obtains, suppose the normal distribution that it is, mean value is 0, and variance is σ ² _ω

1me function in the nlme routine package in R is used for analyzing the data about above model.For each gene, the total dosage effect (H of check in the F-check ₀: β ₁=β ₂=β ₃=β ₄=β ₅=0 with respect to H ₁: at least one β _iBe different).

Although this paper has shown and has described preferred embodiment of the present inventionly that below be tangible for those skilled in the art: these embodiments only are exemplary.In the case of without departing from the present invention, those skilled in the art can carry out many variations, change and substitute.Should be appreciated that, putting into practice when of the present invention, can adopt the various replacement schemes of embodiments of the present invention described herein.Following claim defines scope of the present invention, thereby covers method and structure and equivalent thereof within these claim scopes.

Claims (78)

1. a method of determining the disease of treatment PARP mediation comprises the PARP level in the sample of determining the experimenter, and makes the decision about the disease for the treatment of described PARP mediation, and wherein, described treatment decision is made according to described PARP level.

2. the described method of claim 1, wherein, described treatment decision is about the decision with the PARP modulators for treatment.

3. the method with PARP modulators for treatment disease comprises the PARP level in the sample of determining the experimenter; Make about the decision with PARP modulators for treatment disease, described decision is based on described PARP level; And with the described experimenter's of described PARP modulators for treatment described disease, described treatment determines based on described treatment.

4. each described method in the claim 1 or 3, wherein, described definite PARP level comprises a kind of analytical technology.

5. the described method of claim 4, wherein, described analytical technology is measured the PARP expression of gene.

6. the described method of claim 4, wherein, described analytical technology is measured the PARP-1 expression of gene.

7. the described method of claim 4, wherein, described analytical technology is a polymerase chain reaction.

8. each described method in the claim 1 or 3, wherein, described sample is selected from the preceding liquid of people's normal specimens, tumor sample, hair, blood, cell, tissue, organ, brain tissue, blood, serum, sputum, saliva, blood plasma, nipple aspirated liquid, synovia, celiolymph, sweat, urine, fecal matter, pancreatic juice, girder liquid, celiolymph, tear, bronchial perfusate, swabbing, bronchus aspirated liquid, seminal fluid, prostatic fluid, level in gingival sulcus fluid, vaginal fluids and ejaculation.

9. each described method in the claim 1 or 3, wherein, described PARP level is to adjusted, and described treatment decision is the decision with the described disease of PARP inhibitor for treating.

10. each described method in the claim 1 or 3, wherein, described PARP level is to regulate downwards, and described treatment decision is the decision without the described disease of PARP inhibitor for treating.

11. the described method of each in claim 1 or 3, wherein, described PARP correctives is the PARP inhibitor.

12. the described method of claim 11, wherein, described PARP inhibitor is selected from benzamide, quinolone, isoquinolone, benzopyrone, 3,5-two iodo-4-(4 '-the methoxyl phenoxy group) methyl benzoate and 3,5-two iodo-4-(4 '-methoxyl-3 ', 5 '-two iodo-phenoxy groups) and methyl benzoate, cyclohexyl benzamide, benzimidazole and indoles.

13. the described method of each in claim 1 or 3, wherein, described method further comprises to patient, health care supplier or health care administration person provides conclusion about described disease, and described conclusion is based on described decision.

14. the described method of each in claim 1 or 3, wherein, described treatment be selected from oral administration, mucosal, oral administration, intranasal administration, suction, parenteral, intravenous, subcutaneous, the muscle, hypogloeeis, percutaneous dosing and rectally.

15. the described method of each in claim 1 or 3, wherein, the disease of described PARP mediation is selected from cancer, inflammation, metabolic disease, CVS disease, CNS disease, blood-lymphatic system obstacle, endocrine and neuroendocrine obstacle, urethra obstacle, respiratory system obstacle, female reproductive system obstacle and male reproductive system obstacle.

16. the described method of claim 15, wherein, described cancer is selected from adenocarcinoma of colon, adenocarcinoma of esophagus, hepatocellular carcinoma, squamous cell carcinoma, pancreas adenocarcinoma, islet-cell tumour, rectal adenocarcinoma, GISTs, sdenocarcinoma of stomach, adrenocortical carcinoma, follicular carcinoma, papillary carcinoma, breast cancer, duct carcinoma, lobular carcinoma, comedocarcinoma, mucous carcinoma, phyllodes tumor, adenocarcinoma ovaries, adenocarcinoma of endometrium, GCT, MCAC, adenocarcinoma of cervix, the vulva squamous cell carcinoma, basal-cell carcinoma, adenocarcinoma of the prostate, giant cell tumor of bone, the bone osteosarcoma, laryngocarcinoma, adenocarcinoma of lung, kidney, carcinoma of urinary bladder, wilms' tumor and lymthoma.

17. the described method of claim 15, wherein, described inflammation is selected from non_hodgkin lymphoma, Wegner's granulomatosis, Hashimoto thyroiditis, hepatocellular carcinoma, chronic pancreatitis, rheumatoid arthritis, reactive lymphoid hyperplasia, osteoarthritis, ulcerative colitis and papillary carcinoma.

18. the described method of claim 15, wherein, described metabolic disease is diabetes or obesity.

19. the described method of claim 15, wherein, described CVS disease is selected from atherosclerotic, coronary artery disease, granulomatous myocarditis, chronic myocarditis, miocardial infarction and primary hypertrophic cardiomyopathy.

20. the described method of claim 15, wherein, described CNS disease is selected from Alzheimer disease, cocaine abuse, schizophrenia and Parkinson's.

21. the described method of claim 15, wherein, described blood-lymphatic system obstacle is selected from non_hodgkin lymphoma, chronic lymphocytic leukemia and reactive lymphoid hyperplasia.

22. the described method of claim 15, wherein, described endocrine and neuroendocrine obstacle are selected from nodular hyperplasia, Hashimoto thyroiditis, islet-cell tumour and papillary carcinoma.

23. the described method of claim 15, wherein, described urethra obstacle is selected from clear-cell carcinoma, transitional cell carcinoma and wilms' tumor.

24. the described method of claim 15, wherein, described respiratory system obstacle is selected from gland carcinoma squamosum, squamous cell carcinoma and large cell carcinoma.

25. the described method of claim 15, wherein, described female reproductive system obstacle is selected from gland cancer, liomyoma, MCAC and serous cystadenocarcinoma.

26. the described method of claim 15, wherein, described male reproductive system obstacle is selected from prostate cancer, benign protuberance hyperplasia and seminoma.

27. the described method of each in claim 1 or 3, wherein, described PARP correctives is a 4-iodine, the 3-nitrobenzamide.

28. computer-readable medium that is suitable for transmitting the sample analysis result, it comprises the information by the medicable disease of PARP correctives about the experimenter, described information obtains by following steps: determine the PARP level in described experimenter's the described sample, and make about the decision with the described disease of described PARP modulators for treatment according to described PARP level.

29. each the described method in the claim 1,2 or 28, wherein, at least one step uses a computer and finishes.

30. definite breast cancer can comprise the PARP level in the sample of determining the experimenter with the method for PARP inhibitor for treating, and makes about the whether decision of available described PARP inhibitor for treating of described breast cancer according to described PARP level.

31. method with PARP inhibitor for treating experimenter's breast cancer, comprise the PARP level in the sample of determining described experimenter, make about the whether decision of available described PARP inhibitor for treating of described breast cancer according to described PARP level, and with the described breast cancer of described PARP inhibitor for treating.

32. the described method of each in claim 30 or 31, wherein, described PARP level raises.

33. the described method of claim 32, wherein, described experimenter's BRCA gene has defective.

34. the described method of each in claim 30 or 31, wherein, described experimenter has the BRCA gene of downward modulation.

35. each the described method in the claim 1,2,28,30 or 31, wherein, described PARP is PARP-1.

36. the method to experimenter's breast cancer classification comprises the PARP level in the tumor sample of determining described experimenter, and makes about the decision with the described tumour of PARP modulators for treatment, wherein, described decision is made according to described PARP level.

37. method for the treatment of experimenter's breast cancer, comprise the PARP level in the sample of determining described experimenter, make about decision according to described PARP level with PARP modulators for treatment described tumour, and with the described experimenter's of described PARP modulators for treatment described tumour.

38. the described method of each in claim 36 or 37, wherein, described breast cancer is infitrating ductal carcinoma.

39. the described method of claim 39, wherein, described infitrating ductal carcinoma is ER, Her2-neu and PR feminine gender.

40. the described method of each in claim 36 or 37, wherein, described definite PARP level comprises a kind of analytical technology.

41. the described method of claim 40, wherein, described analytical technology is measured the PARP expression of gene.

42. the described method of each in claim 36 or 37, wherein, described sample is selected from the preceding liquid of human normal specimens, tumor sample, hair, blood, cell, tissue, organ, brain tissue, blood, serum, sputum, saliva, blood plasma, nipple aspirated liquid, synovia, celiolymph, sweat, urine, fecal matter, pancreatic juice, girder liquid, celiolymph, tear, bronchial perfusate, swabbing, bronchus aspirated liquid, seminal fluid, prostatic fluid, level in gingival sulcus fluid, vaginal fluids and ejaculation.

43. the described method of each in claim 36 or 37, wherein, described PARP level is to adjusted.

44. the described method of each in claim 36 or 37, wherein, described PARP correctives is the PARP inhibitor.

45. the described method of claim 44, wherein, described PARP inhibitor is selected from benzamide, quinolone, isoquinolone, benzopyrone, 3,5-two iodo-4-(4 '-the methoxyl phenoxy group) methyl benzoate and 3,5-two iodo-4-(4 '-methoxyl-3 ', 5 '-two iodo-phenoxy groups) and methyl benzoate, cyclohexyl benzamide, benzimidazole and indoles.

46. the described method of each in claim 36 or 37, wherein, described method further comprises to patient, health care supplier or health care administration person provides conclusion about described disease, and described conclusion is based on described decision.

47. the described method of each in claim 36 or 37, wherein, described treatment be selected from oral administration, mucosal, oral administration, intranasal administration, suction, parenteral, intravenous, subcutaneous, the muscle, hypogloeeis, percutaneous dosing and rectally.

48. definite breast cancer can comprise the PARP level in the sample of determining the experimenter, and make about the decision with the described breast cancer of described PARP inhibitor for treating according to described PARP level with the method for PARP inhibitor for treating.

49. method by PARP inhibitor for treating experimenter's breast cancer, comprise the PARP level in the sample of determining described experimenter, make about decision according to described PARP level with PARP inhibitor for treating described breast cancer, and with the described breast cancer of described PARP inhibitor for treating.

50. the described method of each in claim 48 or 49, wherein, described PARP level is to adjusted.

51. the described method of each in claim 48 or 49, wherein, described breast cancer is infitrating ductal carcinoma.

52. the described method of claim 51, wherein, described infitrating ductal carcinoma is ER, Her2-neu and/or PR feminine gender.

53. method for cancer for the treatment of the experimenter, comprise in the cancer sample of determining described experimenter whether having ER, Her2-neu and PR, and with the described cancer of PARP inhibitor for treating, wherein, if described cancer sample is ER, Her2-neu and/or PR feminine gender, then carry out described treatment.

54. a certain stage of the disease of the disease of a definite PARP mediation or PARP mediation can be used the method for PARP modulators for treatment, comprise the PARP level in the sample of determining the experimenter, and determine that described PARP level whether on the level that presets, determines that thus the disease of described PARP mediation can be used the PARP modulators for treatment.

55. one kind by using the method for PARP modulators for treatment disease to the patient, comprise the PARP level in the sample of determining described patient, determine that described PARP level is whether on the level that presets, the disease of definite thus described PARP mediation can be used the PARP modulators for treatment, and by use the described patient's of described PARP modulators for treatment described disease to described patient.

56. the described method of each in claim 54 or 55, wherein, described PARP correctives is the PARP inhibitor.

57. the described method of each in claim 54 or 55, wherein, described PARP is PARP-1.

58. computer-readable medium that is suitable for transmitting the sample analysis result, wherein, described medium comprises the information by the medicable disease of PARP correctives about the experimenter, described information obtains by following steps: determine the PARP level in described experimenter's the described sample, and determine that described PARP level whether on the level that presets, determines that thus the disease of described PARP mediation can be used the PARP modulators for treatment.

59. each the described method in the claim 54,55 or 58, wherein, at least one step uses a computer and finishes.

60. definite breast cancer can comprise the PARP level in the sample of determining the experimenter with the method for PARP inhibitor for treating, and determines that described PARP level whether on the level that presets, determines that thus described breast cancer is medicable with the PARP correctives.

61. method with PARP inhibitor for treating experimenter's breast cancer, comprise the PARP level in the sample of determining described experimenter, determine that described PARP level is whether on the level that presets, determine that thus described breast cancer is medicable with the PARP inhibitor, and by use the described breast cancer of described PARP inhibitor for treating to described patient.

63. claim 60 or 61 described methods, wherein, described experimenter has BRCA1 or BRCA2 defective.

64. the described method of each in claim 60 or 61, wherein, described experimenter has the BRCA expression of gene level of reduction.

65. the described method of each in claim 60 or 61, wherein, described PARP is PARP-1.

66. the method to patient's breast cancer classification comprises the PARP level in the tumor sample of determining described patient, and determines described PARP level whether on the level that presets, thus with described breast cancer classification for medicable with the PARP correctives.

67. method for the treatment of experimenter's breast cancer, comprise the PARP level in the sample of determining described experimenter, determine that described PARP level is whether on the level that presets, determine that thus described breast cancer is medicable with the PARP correctives, and with the described patient's of described PARP modulators for treatment described tumour.

68. the described method of each in claim 66 or 67, wherein, described breast cancer is infitrating ductal carcinoma.

69. the described method of claim 68, wherein, described infitrating ductal carcinoma is ER, Her2-neu and/or PR feminine gender.

70. the described method of each in claim 66 or 67, wherein, described PARP correctives is the PARP inhibitor.

71. definite breast cancer can comprise the PARP level in the sample of determining the patient with the method for PARP inhibitor for treating, and determines that described PARP level whether on the level that presets, determines that thus described breast cancer is medicable with the PARP inhibitor.

72. method with PARP inhibitor for treating patient's breast cancer, comprise the PARP level in the sample of determining described patient, determine that described PARP level is whether on the level that presets, determine that thus described breast cancer is medicable with the PARP inhibitor, and by use the described breast cancer of described PARP inhibitor for treating to described patient.

73. the described method of each in claim 71 or 72, wherein, described breast cancer is infitrating ductal carcinoma.

74. the described method of claim 73, wherein, described infitrating ductal carcinoma is ER, Her2-neu and/or PR feminine gender.

75. method for cancer for the treatment of the patient, comprise in the cancer sample of determining described patient and whether have ER, Her2-neu and/or PR, and when ER, Her2-neu and/or PR are not present in described patient's the described sample, with the described cancer of PARP inhibitor for treating.

76. the method for PARP inhibitor for treating selection experimenter comprises:

Before using the PARP inhibitor, measure the PARP level from the biological sample that the experimenter collects, determine that the PARP level in the sample is higher than preset value, and select this experimenter to be used for the PARP inhibitor for treating.

77. the method with PARP inhibitor for treating experimenter comprises:

Before using the PARP inhibitor, measure the PARP level from the biological sample that the experimenter collects, determine that the PARP level in the sample is higher than preset value, and use the PARP inhibitor to this experimenter.

78. the method for the experimenter of PARP inhibitor for treating for the response of treatment accepted in an evaluation, this method comprises: at least in the very first time point and the second time point determining experimenter's PARP level, to obtain a PARP level and the 2nd PARP level at least, wherein, the 2nd PARP level reduces the positive response of indication to treatment than a PARP level.

79. its situation of treatment causes the patient's of PARP level rising method, wherein, the PARP level in patient's sample is higher than default PARP level, and this method comprises the PARP inhibitor of administering therapeutic effective dose.

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